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Cardiovascular Research 1998 37(1):179-186; doi:10.1016/S0008-6363(97)00203-4
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Selective impairment of HCO3–-dependent pHi regulation by lysophosphatidylcholine in guinea pig ventricular myocardium

Seiichi Yamaguchi, Masaji Tamagawa, Nobuyuki Nakajima and Haruaki Nakaya*

Department of Pharmacology and First Department of Surgery, Chiba University School of Medicine, Chiba 260, Japan

* Corresponding author. Tel. (+81-43) 2262050; Fax (+81-43) 2262052, E-mail: nakaya@med.m.chiba-u.ac.jp

Received 12 February 1997; accepted 30 July 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The aim was to examine the effects of lysophosphatidylcholine (LPC), an amphiphilic lipid metabolite in ischemic myocardium, on intracellular pH (pHi) regulatory systems in guinea pig papillary muscles. Methods: In CO2/HCO3–-buffered Tyrode solution, pHi, intracellular Na+ activity (aNai) and membrane potential of isolated guinea pig papillary muscles were measured using ion-selective microelectrode and conventional microelectrode. Standard ammonium prepulsing with 20 mM NH4Cl was used to produce an intracellular acid load, and effects of LPC on the pHi recovery from acidosis were evaluated in the absence and presence of a transport inhibitor. Results: LPC acidified the resting pHi by 0.03±0.01 pH units (n = 15, P<0.01) concomitantly with a slight decrease in resting membrane potential and an increase in aNai in quiescent preparations. The pHi recovery rate from an intracellular acid load was decreased to 83±4% of the control value by 30 µM LPC (n = 8, P<0.05) but not by 30 µM phosphatidylcholine (PC). In the presence of 10 µM 5-(N,N–hexamethylene) amiloride (HMA), a Na+–H+ exchange inhibitor, LPC still slowed pHi recovery from an intracellular acid load to 77±4% of the control (n = 5, P<0.05). However, LPC failed to alter the pHi recovery rate in the presence of 4,4'-diisothiocyanatostilbene–2,2'-disulfonic acid (DIDS, 0.5 mM), a Na+-HCO3– symport inhibitor. Conclusion: LPC impairs Na+–HCO3– symport but not Na+–H+ exchange, and LPC may potentiate its arrhythmogenic action by intensifying the intracellular acidosis in ischemic myocardium.

KEYWORDS Lysophosphatidylcholine; Guinea pig papillary muscles; Intracellular pH; Intracellular Na+ activity; Na+–H+ exchange; Na+–HCO3– symport; Ion-selective microelectrode


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Lysophosphatidylcholine (LPC) is an amphiphilic lipid metabolite which accumulates in ischemic myocardium [1, 2]. LPC is considered to play a role in the genesis of ischemia-induced arrhythmias. In isolated cardiac tissues LPC decreases the maximum diastolic potential and the action potential duration and induces abnormal automaticities such as afterpotentials and rhythmic automatic depolarizations [3–6]. LPC has been shown to affect a number of cardiac ion channels including Na+ channels [7–9]and K+ channels [10–12]. In addition, LPC is known to inhibit cardiac sarcolemmal Na+–K+ ATPase [13, 14]. However, it remains unclear whether LPC affects the intracellular pH (pHi) regulatory systems in ventricular myocardium. It has been acknowledged that several sarcolemmal ion carriers, such as Na+–H+ exchange, Na+-HCO3– symport and Cl––HCO3– exchange, are involved in the regulation of pHi in heart cells [15–18]. In this study, effects of LPC on the pHi regulatory systems were examined in guinea pig ventricular myocardium by using ion-selective microelectrode technique. The findings presented here indicate that LPC impairs HCO3–-dependent pHi regulation but not Na+–H+ exchange system in ventricular myocardium. A preliminary account of this study has appeared in an abstract form [19].


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Tissue preparations and solutions
All experiments were performed under the regulations of the Animal Research Committee of Chiba University School of Medicine. This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). Guinea pigs weighing 180–400 g were stunned with a blow on the head, and their hearts were quickly removed. The hearts were immersed in oxygenated Tyrode solution, and papillary muscles less than 1mm in diameter were carefully dissected under a microscope from the right ventricle of the hearts. The preparations were transferred to a tissue bath of 5 ml volume and superfused at a rate of 10 ml/min with modified Tyrode solution containing the following composition (in mM): NaCl 125, KCl 4, NaHCO3 25, NaH2PO4 1.8, MgCl2 0.5, CaCl2 1.8 and glucose 5.5. The solution was gassed with 95% O2+5% CO2 and the temperature was kept constant at 36.0±0.5°C. During an equilibration period the preparations were electrically stimulated at 0.5 Hz through platinum field electrodes. Stimuli were rectangular pulses of 1 ms duration at twice the diastolic threshold, delivered from an electronic stimulator (Nihon Kohden S-7272B, Tokyo, Japan).

2.2 Electrical arrangements
Transmembrane potentials were recorded with conventional microelectrodes pulled from filamented borosilicate tubing (o.d. 2.0 mm, i.d. 1.15 mm; Narishige Scientific Instrument Lab, Tokyo, Japan) and filled with 3 M KCl (resistance 10–30 M{Omega}). In these experiments the developed tension of the papillary muscles was also measured concomitantly. One end of the preparation was hooked to the lever arm of a force transducer (Nihon Kohden, TB 651T, Tokyo, Japan) mounted on a micromanipulator and the other was pinned to the bottom of the tissue chamber. Resting tension was progressively increased to 2 mN.

Single-barrelled pH- and sodium-selective microelectrodes were used to measure intracellular pH (pHi) and sodium ion activities (aNai) as the differential signal between an intracellular conventional microelectrode and an ion selective microelectrode, as previously described [20]. The conventional microelectrode was coupled via an Ag/AgCl junction to a high-input impedance amplifier (W-P Instruments, FD-223, New Haven, CT, USA). The ion-selective electrode was also connected to the input stage of the same electrometer. An agar bridge containing 3 M KCl was used as a common reference electrode. The amplified signals were displayed on a dual beam oscilloscope (Nihon Kohden, VC-11, Tokyo, Japan) and recorded on a chart recorder (Watanabe Sokki, WR-3101, Tokyo, Japan).

Hydrogen ion selective electrodes (H-ISE) were siliconized in the oven (200°C) with 100 µl of siloxane (Shin-Etsu Chemical Co., Tokyo, Japan) for 2 h and bevelled to a tip size of <1.0 µm. A short column of hydrogen ionophore-cocktail (Fluka 95291, Buchs SG, Switzerland) was drawn up into the tip of the electrode by applying negative pressure, and then the electrode was back-filled with 100 mM–NaCl and 50 mM–HEPES–NaOH (pH 7.4 at 20°C). The H-ISEs were calibrated by using calibration solutions of a composition which mimicked the intracellular environment (KCl 140, NaCl 10, MgCl2 0.5, HEPES-KOH 5 with pH of 6.4, 6.9, 7.4 and 7.9). Acceptable electrodes displayed a sensitivity of 56–61 mV/pH units. Sodium ion selective electrodes (Na-ISE) were pulled and siliconized in the same manner as H-ISE, drawn up a short column of liquid-sensor cocktail for Na+ (Fluka, 71176) into the tip by applying negative pressure, and then back-filled with 500 mM NaCl solution. The slope of the Na-ISE response (SNa) was 27.7±0.3 mV/e-fold increase in sodium ion activities (63.5±0.6 mV/tenfold increase), and the selectivity coefficient (kNa/K) ranged from 0.016 to 0.042 (0.034±0.004). In order to keep stable impalements with two electrodes (conventional and ion selective electrodes) in the same preparation long enough to accomplish each experiment, electrical stimulation was ceased after the attainment of a stable impalement.

2.3 Calculation of sarcolemmal acid efflux and measurement of aNai
Standard ammonium prepulsing of 5–10 min with 20 mM NH4Cl in bicarbonate-buffered solution was used to decrease pHi. After the introduction of NH4Cl, NH3 rapidly diffuses into the cell, where most of it combines with H+, resulting in a rise in pHi. This is followed by a slower passive entry of NH4+, leading to slow reacidification. Removal of NH4Cl then causes a large intracellular acidification since all the accumulated NH4+ diffuses out of the cell as NH3, leaving H+ behind [21].

We calculated net acid efflux (JHe) following an acid load using the following equation:


Formula

where βT is the total intracellular buffering power, dpHi/dt is the rate of pHi recovery at any given pHi and {delta} is the background rate of acid loading in the absence of any acid extrusion [22]. The term βT comprises the sum of intrinsic buffering power (βi) plus buffering power caused by intracellular CO2/HCO3– (βCO2). The efflux through Na+–H+ exchange was estimated from pHi recovery following an acid load in HCO3– buffered solution in the presence of 0.5 mM 4,4'-diisothiocyanatostilbene–2,2'-disulfonic acid (DIDS). The efflux through Na+–HCO3– symport was estimated from pHi recovery occurring in HCO3–-buffered solution in the presence of 10 µM 5-(N,N-hexamethylene) amiloride (HMA). Under these conditions, βT=βi+βCO2, with βi=–28pHi+222.6 [23]and βCO2=2.3[HCO3–]i, where, from the Henderson-Hesselbalch equation, the value of [HCO3–]i is:


Formula

The constant background intracellular acid loading rate ({delta}), due to primarily to the de novo production of metabolic acid within the cell, was considered to be 0.4±0.06 mEq/l/min in HEPES-buffered Tyrode solution [23]. In addition, it was considered that background acid loading in CO2/HCO3–-buffered solution would be similar to that which had been determined previously in HEPES-buffered solution, at least when pHi was reduced [18]. For simplicity, we assumed the background acid loading to be a constant.

It was confirmed that the pHi recovery rate from an intracellular acid load induced by the NH4Cl prepulse method was reproducible in 6 preparations when the acid load was repeated in the absence of lysophosphatidylcholine or phosphatidylcholine. In order to inhibit Na+–H+ exchange system, HMA (10 µM), an amiloride analog which is more potent than amiloride [24], was used. As an inhibitor of Na+–HCO3– symport, DIDS (0.5 mM) was used although the compound is known to inhibit Cl––HCO3– exchange and Cl– channels [16, 17, 25]. We could not use a HCO3–-free buffer solution to examine the involvement of Na+–HCO3– symport because the use of HEPES-buffered solution appeared to be somewhat harmful to the isolated preparations and the maintenance of impalement with both electrodes for long time was impossible.

The aNai was determined from a calibration curve, which was constructed using mixed solutions of NaCl and KCl with a constant ionic strength (150 mM). Ion activity coefficients for calibrating single electrolyte solutions were taken from Shedlovsky [26], and those for calibrating mixed solution were calculated using the equation of Pitzer and Mayorga [27].

2.4 Chemicals
The following chemicals were used: 5–(N,N–hexamethylene) amiloride (HMA) and 4,4'–diisothiocyanatostilbene–2,2'–disulfonic acid (DIDS) (Sigma Chemical, St. Louis, MO, USA), L–{alpha}–lysophosphatidylcholine palmitoyl (LPC) and phosphatidylcholine palmitoyl (PC) (Wako Pure Chemical Industries, Osaka, Japan). LPC or PC was dissolved in chloroform, and appropriate aliquots of the solution were dried with a stream of N2 gas, followed by sonication in the Tyrode solution.

2.5 Statistics
All data are presented as mean±s.e.m. Statistical analyses of the data were performed using Wilcoxon test. P-values less than 0.05 were considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effects of LPC on membrane potential and twitch tension
After exposure to 30 µM LPC for 30 min the resting membrane potential (RMP) slightly but significantly decreased by 0.5±0.2 mV from the control value (n = 13, P<0.05). The baseline values of RMP, action potential amplitude (APA), action potential duration at 50% (APD50) and 90% repolarization level (APD90), the maximum upstroke velocity of phase 0 depolarization (Vmax) and twitch tension (TT) of 6 papillary muscles stimulated at 0.5 Hz were –95.7±1.4 mV, 129.2±0.9 mV, 170.1±8.3 ms, 201.1±11.4 ms, 244.6±17.4 V/s and 13.8±1.0 mg, respectively. LPC at a concentration of 30 µM increased TT by 59±19% (n = 6, P<0.05) although it did not significantly change APD90, APA and Vmax. LPC at a higher concentration of 100 µM decreased APD90, RMP and Vmax to 96±2%, 95±4% and 97±2% of the control, respectively (n = 3).

3.2 Effects of LPC on pHi and aNai
The baseline pHi value was 7.21±0.01 in quiescent papillary muscles (n = 33). LPC at a concentration of 30 µM slightly but significantly acidified the resting pHi by 0.03±0.01 pH units in CO2/HCO3––buffered Tyrode solution (n = 17, P<0.01), as shown in Fig. 1a. These results imply that LPC may alter the function of pHi regulatory system(s) in sarcolemma because LPC per se did not affect the pH of the Tyrode solution.


Figure 1
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  		Fig. 1 Effect of LPC (30 µM) on pHi recovery from an intracellular acidification. (a): Actual record of pHi recovery from an intracellular acid load induced by the NH4+ pre-pulse method (20 mM NH4Cl) in HCO3–-buffered solution in the absence (left) and presence of 30 µM LPC (right). (b): Total net acid efflux (JHe, see Materials and Methods) is plotted versus pHi in the absence (open circles) and presence (filled circles) of 30 µM LPC. The data were obtained from the experiment shown in (a).

 
Effects of LPC on the pHi recovery from an intracellular acid load induced by the NH4Cl (20 mM) prepulse method [21]were examined in guinea pig papillary muscles, as shown in Fig. 1a. Recovery from an acid load in bicarbonate-buffered solution was inhibited by the presence of 30 µM LPC. Fig. 1b plots acid efflux as a function of pHi, and the relationship was shifted to the left by LPC along the pHi axis. Efflux data was calculated from the pHi recoveries in the absence and presence of LPC. In a total of 8 preparations, net acid efflux at pHi of 7.00 was found to be 2.14±0.15 and 1.80±0.18 mEq/l/min in the absence and presence of 30 µM LPC, respectively. Inhibitory effect of LPC on acid efflux was concentration-dependent although LPC significantly decreased the net acid efflux only at a concentration of 30 µM (Fig. 2). The net acid efflux was changed from 1.78±0.26 to 1.78±0.37 mEq/l/min after 3 µM LPC (n = 4, N.S.) and from 1.93±0.30 to 1.84±0.32 mEq/l/min after 10 µM LPC (n = 5, N.S.). Phosphatidylcholine (PC) at a concentration of 30 µM did not inhibit the pHi recovery from an intracellular acid load. The net acid efflux at pHi of 7.00 was 1.83±0.24 and 2.01±0.29 mEq/l/min before and after 30 µM PC, respectively (n = 5, N.S.).


Figure 2
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  		Fig. 2 Effects of LPC or phosphatidylcholine (PC) on pHi recovery from an intracellular acidification. The normalized net acid efflux calculated at pHi of 7.00 is indicated on the ordinate. Values are expressed as mean±s.e.m. *P<0.05 vs. Control.

 
It is known that in cardiac cells the Na+–H+ exchange and HCO3–-dependent pHi regulatory systems, potentially Na+–HCO3– symport, are important for the extrusion of hydrogen ions [18]. The former is blocked by the amiloride analogue HMA and the latter is inhibited by DIDS [17, 23]. In order to determine which pHi regulatory system is affected by LPC, we evaluated the effects of LPC on pHi recovery from an intracellular acid load in the presence of one of these transport inhibitors. In part of experiments influences of HMA and DIDS on the recovery from an acid load were examined in the bicarbonate-buffered solution. Treatment with 10 µM HMA decreased the acid efflux at pHi of 7.00 from 2.88±0.52 to 2.18±0.20 mEq/l/min in 4 preparations. In other 4 preparations DIDS (0.5 mM) also decreased the acid efflux from 2.27±0.18 to 1.93±0.27 mEq/l/min. Thus, HMA and DIDS per se decreased the net acid efflux by 20.3±7.6% and by 17.3±5.7%, respectively.

In the presence of 10 µM HMA, LPC at a concentration of 30 µM markedly slowed pHi recovery from an intracellular acid load, as shown in Fig. 3. The pHi dependence of HMA-resistant acid extrusion, which was probably due to Na+–HCO3– symport, was shifted to the left by LPC. In the presence of 0.5 mM DIDS, the pHi recovery was considered to be due solely to Na+–H+ exchange since HCO3–-dependent pHi regulatory systems are inhibited by DIDS [17]. In this condition, however, LPC hardly affected the pHi dependence of acid extrusion, as shown in Fig. 3.


Figure 3
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  		Fig. 3 Effect of LPC (30 µM) on pHi recovery from intracellular acidification in the presence of 10 µM HMA or 0.5 mM DIDS. (a): Actual record of the control recovery from an acid load in HCO3–-buffered solution containing HMA (left) and the recovery in the presence of HMA and LPC (right). (b): Actual record of control recovery from an acid load in HCO3–-buffered solution containing DIDS (left) and the recovery in the presence of DIDS and LPC (right). Net acid efflux (JHe) versus pHi obtained from (a) and (b) are shown in (c) and (d), respectively. Note that LPC inhibited the acid efflux in the presence of HMA but not in the presence of DIDS.

 
Fig. 4 summarizes the changes in net acid efflux at a test pHi of 7.00 after 30 µM LPC in the absence and presence of HMA or DIDS. LPC significantly decreased the net acid efflux by 16.8±3.6% from the control value of 2.14±0.15 mEq/l/min in the absence of any transport inhibitor (n = 8, P<0.05). LPC also significantly decreased the net acid efflux at pHi of 7.00 from 2.04±0.24 to 1.56±0.18 mEq/l/min (22.7±4.0% decrease) in the presence of HMA, a Na+–H+ exchange blocker (n = 5, P<0.05). In the presence of DIDS, however, LPC failed to decrease the net acid efflux. In DIDS treated preparations the acid efflux at pHi of 7.00 before and after 30 µM LPC was 1.83±0.27 and 1.90±0.33 mEq/l/min, respectively (n = 6, N.S.). These findings suggest that LPC impairs HCO3–-dependent pHi regulatory system but not Na+–H+ exchange in guinea pig ventricular myocardium.


Figure 4
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  		Fig. 4 Summarized data of net acid efflux estimated at pHi of 7.00 in HCO3–-buffered solution. The open and solid columns represent net acid efflux in the absence (control) and presence of 30 µM LPC. Values are expressed as mean±s.e.m. of 5 to 8 experiments. *P<0.05 vs. Control. Note that LPC significantly reduced the net acid efflux in the absence and presence of HMA but not in the presence of DIDS.

 
An increase in intracellular Na+ might indirectly affect pHi by inhibiting Na+–HCO3– symport. Therefore, we examined effects of 30 µM LPC on aNai in the same experimental condition. A representative recording of change in intracellular Na+ activity (aNai) after the exposure to 30 µM LPC is illustrated in Fig. 5a and summarized in Fig. 5b. After the introduction of 30 µM LPC, aNai was significantly increased from 6.9±1.0 to 8.0±1.2 mM in 8 quiescent preparations (P<0.05).


Figure 5
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  		Fig. 5 Effects of LPC (30 µM) on intracellular Na+ activity (aNai) in guinea pig papillary muscles. (a): Actual record of change of aNai after exposure to 30 µM LPC. (b): Summarized data of aNai change after 30 µM LPC in 8 preparations. Values are expressed as mean±s.e.m. *P<0.05 vs. Control.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
LPC has been shown to accumulate within minutes after the onset of myocardial ischemia in vivo [28, 29]. Studies using electron microscopic autoradiography have indicated that electrophysiological abnormalities are manifested when LPC is present in the sarcolemmal membrane at a concentration of 2 mole% [30, 31]. This concentration of LPC in the sarcolemma was produced by exposure of isolated cardiac tissues to 100 µM LPC. In addition, the concentration of LPC has been reported to increase dramatically in the venous effluent from ischemic myocardium and in cardiac lymph of experimental animals [32, 33]. The calculated concentration of LPC in extracellular space is expected to reach about 200 µM after 15 minutes of ischemia. These concentrations of LPC are sufficient to induce electrophysiological abnormalities in vitro and in vivo [33–35].

LPC has been reported to produce marked alterations in transmembrane action potentials of isolated cardiac tissues in concentrations higher than 30 µM [2, 3, 6]. The electrophysiological derangements might be attributable to dysfunction of the cardiac ion channels. LPC has been shown not only to decrease the peak Na+ current [9]but also to delay the inactivation of the sodium channels [7]. LPC has also been reported to inhibit the rectifier K+ channels [10, 11]. The LPC-induced ion channel dysfunction might lead to the resting membrane depolarization and the slowing in conduction in ischemic myocardium. Consistent with the previous reports [2, 3, 6], LPC at a concentration of 30 µM slightly but significantly depolarized the resting membrane and shortened APD at a higher concentration (100 µM).

In terms of sarcolemmal ion transport, it has been reported that LPC inhibits Na+–K+ ATPase activity in relatively low concentrations (10–30 µM) [13, 14]. Depression of Na+–K+ pump results in an increase in [Na+]i, which is then followed by an increase in [Ca2+]i through the Na+–Ca2+ exchange mechanism. The increase in [Ca2+]i may be responsible for delayed afterdepolarizations observed LPC-treated Purkinje fibers [5]. In the present study LPC at a concentration of 30 µM increased twitch tension, although LPC produced a slight intracellular acidification, which is expected to decrease the twitch tension by lessening the Ca2+-sensitivity of the myofilaments [36]. In the present study, LPC significantly increased aNai in quiescent preparations. The increase in aNai might be attributable to the inhibition of Na+–K+ pump [13, 14]and/or activation of nonspecific cation channels [37]. A similar increase in aNai after exposure to LPC was also observed in isolated arterially perfused rabbit papillary muscles [38]. An increase in [Na+]i might lead to an intracellular Ca2+ overload through Na+–Ca2+ exchange.

To maintain physiological pHi, the cardiac cells possess at least two alkalinizing ionic transporters, a Na+–H+ exchange and a Na+–HCO3– symport, and one acidifying Cl––HCO3– exchange [15, 22, 39]. In the present study, the Na+–H+ antiport blocker HMA decreased the acid extrusion rate at pHi of 7.00 by 20%, while the Na+–HCO3– symport blocker DIDS also decreased it by 17% in guinea pig papillary muscles. One of these transporters might compensate for the decrease in the acid extrusion capacity when the other transporter was pharmacologically blocked. LPC at a concentration of 30 µM acidified the resting pHi and decreased the acid efflux rate approximately by 20%. Therefore, LPC is considered to inhibit Na+–H+ exchange and/or a Na+–HCO3– symport. The slowing of pHi recovery from an intracellular acid load by LPC was observed in the presence of HMA but not DIDS, suggesting that LPC may selectively impair the Na+–HCO3– symport. The inhibitory action of LPC on pHi recovery from an acid load was concentration-dependent although only 30 µM LPC significantly slowed the pHi recovery. We could not examine the influence of higher concentrations of LPC because high concentrations of LPC depolarized the resting membrane potential rapidly and made it difficult to keep stable impalements with both electrodes for enough time. Since PC failed to affect the pHi recovery, the inhibitory effect of LPC on the pHi regulation would not be nonspecific as an amphiphilic compound. It is not clear from the present study why LPC selectively affected the function of Na+–HCO3– symport. One explanation may be that LPC might bind to Na+–HCO3– symport protein more readily than to Na+–H+ antiport protein. Another possibility is that Na+–HCO3– symport might be more susceptible to pertubation of the phospholipid bilayer by incorporation of LPC into the sarcolemma than Na+–H+ antiport. It is also conceivable that Na+–HCO3– symport might be more sensitive to a LPC-induced increase in [Na+]i than Na+–H+ antiport. Indeed, the energy for acid extrusion via the Na+–HCO3– symport system is derived from the transmembrane gradients for Na+ and HCO3– [23]. However, the dependence of the symport upon [Na+]i has not been examined systematically although the dependence on [Na+]o was documented convincingly [23]. Whatever the mechanism(s) involved, the present results show that LPC produces intracellular acidification by inhibiting the Na+–HCO3– symport.

It may be important to think about the pathophysiological implication of LPC-induced intracellular acidosis although the magnitude of the acidosis may be small. Electrophysiological derangements induced by LPC are potentiated at acid pH [32, 34]. Furthermore, membrane-bound lysophospholipase, an enzyme mediating LPC catabolism, is inhibited at acid pH, resulting in accumulation of LPC [2]. Therefore, LPC may intensify the arrhythmogenic action by its own acidifying action.

It is well-known that the inhibition of Na+–H+ antiport produces cardioprotection during myocardial ischemia and reperfusion [40, 41]. At the time of reperfusion, a large outwardly-directed gradient of protons is produced across the sarcolemma, which activates the Na+–H+ antiport and produces an increase in [Na+]i. The increase in [Na+]i leads to an intracellular Ca2+ overload, myocardial dysfunction and arrhythmias [40, 41]. It has been also proposed that the Na+–HCO3– symport may be also activated and contribute to the intracellular Na+ overload during ischemia and reperfusion [42]. The inhibition of the Na+–HCO3– symport by LPC, observed in this study, could lessen the intracellular Na+ overload. However, LPC failed to inhibit the Na+–H+ antiport in the present study. Moreover, LPC increased aNai probably by inhibiting Na+–K+ ATPase [13, 14]and/or activating nonspecific cation channels [37]. Therefore, it can not be expected that LPC affords cardioprotection during ischemia and reperfusion.

Recently LPC has been implicated as a mediator of endothelial dysfunction [43, 44]. LPC that accumulates in oxidized low-density lipoprotein (LDL) impairs endothelium-dependent arterial relaxation through the reduction of endothelium-derived relaxing factor (NO) and/or endothelium-derived hyperpolarizing factor (EDHF) [45–47]. It has been shown that pHi regulatory systems including Na+–H+ exchange, Na+-dependent HCO3––Cl– exchange and Na+–HCO3–-symport exist in vascular smooth muscles and endothelial cells [48, 49]. LPC may modulate the functions of smooth muscle and endothelial cells by influencing the pHi regulatory system.

In conclusion, LPC impairs Na+–HCO3– symport but not Na+–H+ exchange in ventricular myocardium. LPC may potentiate the arrhythmogenic action by intensifying the intracellular acidosis in ischemic myocardium.

Time for primary review 40 days.


   Acknowledgements
 
We are grateful to Prof. L. Kirlin for reading the manuscript. We also thank I. Sakashita for secretarial assistance.


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

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