Introduction

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Lysophosphatidylcholine is the major lysophospholipid in mammalian tissues (Man et al., 1990), and is formed from phosphatidylcholine by the action of phospholipase A₂. An increase in the level of cardiac LPC because of ischemia is thought to be one of the mechanisms underlying the pathogenesis of ischemic injury (Sedlis et al., 1988, 1993). Recently we demonstrated that the accumulation of this lysophospholipid in the SL membrane under ischemic conditions may diminish the availability of phosphatidylinositol 4,5-bisphosphate for the production of second messengers upon the activation of phospholipase C (Liu et al., 1997). It should be pointed out that LPC has toxic effects on the myocardium and these include electrophysiological disturbances like shortened refractory period, decreased membrane potential, increased membrane resistance as well as arrhythmogenesis (Clarkson and Ten Eick, 1983; Corr et al., 1982; Pogwizd et al., 1986), and mechanical alterations such as decreased contractility as well as contracture in cardiac myocytes and perfused hearts (Woodley et al., 1991; Ver Donck et al., 1992; Hoque et al., 1995, 1997). Although LPC has been shown to increase the [Ca⁺⁺]_i in the heart (Corr et al., 1982; Sedlis et al., 1983; Karli et al., 1979), the mechanisms proposed for this effect are not conclusive. In this regard, some investigators have suggested increased SL permeability (Corr et al., 1982) and increased Ca⁺⁺ influx through the L-type voltage-gated Ca⁺⁺ channels (Sedlis et al., 1983), whereas others have indicated inhibition of the Na⁺-K⁺ ATPase activity (Karli et al., 1979) to explain the LPC-induced increase in [Ca⁺⁺]_i. In the present study we investigated the effect of exogenous LPC on rat adult cardiomyocytes and probed the mechanisms responsible for the LPC-induced increase in [Ca⁺⁺]_i by use of some inhibitors of the SL and SR membrane proteins which are involved in the regulation of [Ca⁺⁺]_i. Our results indicate that LPC increases [Ca⁺⁺]_i in a dose-dependent manner, and this increase depends on the entry of extracellular Ca⁺⁺ via the Na⁺-Ca⁺⁺ exchanger in the SL membrane as well as on the status of intracellular SR Ca⁺⁺ stores.

	Methods

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Chemicals. L--Lysophosphatidylcholine, amiloride, ouabain, ryanodine, thapsigargin, EGTA, and BSA were purchased from the Sigma Chemical Company (St. Louis, MO). Collagenase (type II, 310 U/mg) was from the Worthington Biochemicals (Freehold, NJ). Fura-2/AM was obtained from the Molecular Probes (Eugene, OR). All other substances were of analytical purity and purchased from Mallinckrodt (Montreal, Quebec, Canada).

Isolation of cardiomyocytes. Cardiomyocytes were isolated from rat hearts by a collagenase digestion procedure (Xu et al., 1996). Male Sprague-Dawley rats, weighing 300 to 350 g, were injected with heparin (1000 U/100 g b.wt.) and anesthetized with xylazine (10 mg/kg) combined with ketamine (60 mg/kg). After the rats were anesthetized, the heart was excised and cannulated on a Langendorff apparatus via the aorta and perfused in a noncirculatory manner with Ca⁺⁺-free buffer containing (mM): NaCl, 90; KCl, 10; KH₂PO₄, 1.2; MgSO₄, 5; NaHCO₃, 15; taurine, 30; glucose, 20 (pH 7.4); this medium was gassed with 95% O₂ and 5% CO₂ mixture. After 5 min of perfusion, the heart was switched to the same medium containing 0.04% collagenase and 50 µM CaCl₂. At the end of a 30-min recirculation period, the heart was taken off the cannula and the atria were excised. The ventricles were minced into small pieces and subjected to another 30 min of digestion in a fresh collagenase solution in the presence of 1% BSA gassed with 95% O₂ and 5% CO₂ on a shaking water bath at 37°C. Ventricular fragments were triturated gently with a glass Pasteur pipette (once every minute). The cell suspension was collected every 10 min and filtered through a 200 µM nylon mesh. The myocytes were resuspended in buffers, where the extracellular calcium concentration was increased in a gradual stepwise fashion (250 µM, 500 µM, 750 µM, 1000 µM); the cells were incubated in each calcium concentration for 15 min at room temperature. The rod-shaped myocytes comprised more than 80% of the final cell population.

Measurement of fluorescence. The technique for monitoring [Ca⁺⁺]_i with Fura-2/AM as the fluorescent dye has been described previously (Xu et al., 1996). Freshly isolated adult rat cardiomyocytes were incubated with 5 µM Fura-2/AM for 40 min in Krebs-Henseleit buffer containing (mM): NaCl, 90; KCl, 10; KH₂PO₄, 1.2; MgSO₄, 5; NaHCO₃, 15; glucose, 20 (pH 7.4), then washed three times to remove any extracellular dye. The cell number in the cuvette was adjusted to 0.3 million cells/ml. The alteration of the intensity of fluorescence was monitored by a SLM DMX-1100 dual wavelength spectrofluorometer. The excitation wavelength was 340/380 nm, the emission wavelength was 510 nm. The integration time was 0.95 sec; the resolution time was 1.0 sec. The [Ca⁺⁺]_i levels were calculated according to the formula: The ratio (R) of 340/380 nm was calculated automatically. R_max and R_min values were determined by addition of 20 µl Triton X-100 (10%) and 40 µl EGTA (400 mM) respectively. Sf₂ and Sb₂ are the fluorescence proportionality coefficients obtained at 380 nm (excitation wavelength) under R_min and R_max conditions, respectively.

Experimental protocol. The Fura-2/AM-loaded cells were exposed to LPC in the range of 2.5 to 10 µM (final concentration), and the response was traced for 5 min. EGTA (1 mM) was used to remove the extracellular Ca⁺⁺ from the suspension containing the cells in a set of experiments. A group of experiments involved the incubation of myocytes for 10 min with inhibiting agents, namely: a) verapamil, a SL Ca⁺⁺ channel blocker; b) ouabain, an inhibitor of SL Na⁺-K⁺ ATPase; c) amiloride and Ni⁺⁺, inhibitors of SL Na⁺-Ca⁺⁺ exchanger; d) thapsigargin, a SR Ca⁺⁺-pump ATPase inhibitor; and e) ryanodine at low micromolar concentrations, a SR Ca⁺⁺-release channel stimulator. These treated cells subsequently were exposed to 10 µM LPC for 5 min. In another set of experiments, Fura-2/AM-loaded myocytes were incubated for 10 min with a combination of inhibitors, amiloride plus thapsigargin and amiloride plus ryanodine. These treated cells subsequently were exposed to 10 µM LPC.

Data analysis. All results are expressed as mean ± S.E. The increment of [Ca⁺⁺]_i was calculated on the basis of net increase above basal value in each experiment. Increase in [Ca⁺⁺]_i within the groups was assessed by calculating the peak and plateau [Ca⁺⁺]_i. Differences in basal values within groups also were calculated. Statistical analysis was performed by one-way analysis of variance. P values less than .05 reflected significant differences between control and experimental groups.

	Results

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The effects of varying concentrations of LPC on the [Ca⁺⁺]_i in cardiomyocytes were studied. Figure 1 shows a concentration-dependent increase in [Ca⁺⁺]_i by LPC; 10 µM LPC elicited the maximum increase in [Ca⁺⁺]_i. Under the experimental conditions used in this study, 10 µM LPC had no significant effect on cell viability. In fact, the percentage of rod-shaped cells in the absence or presence of 10 µM LPC was 87 ± 0.7 or 87.1 ± 1.1, respectively. Furthermore, the lactate dehydrogenase activity was not detectable in the culture medium under both the above-mentioned conditions. To test whether the increase in [Ca⁺⁺]_i induced by LPC depended on Ca⁺⁺-influx, the relationship between extracellular calcium concentration and LPC-induced increase in [Ca⁺⁺]_i was investigated. As shown in figure 2, the LPC-induced increase of [Ca⁺⁺]_i above the basal value was proportional to the concentration of Ca⁺⁺ in the extracellular medium. In addition, the increase in [Ca⁺⁺]_i caused by LPC was abolished by chelating Ca⁺⁺ in the medium with 1 mM EGTA. To assess the role of L-type Ca⁺⁺ channel as a possible mechanism involved in the LPC-evoked entry of Ca⁺⁺, the effect of verapamil, a specific blocker of the L-type Ca⁺⁺ channel (Lee and Tsien, 1983) on the LPC-induced [Ca⁺⁺]_i increase, was studied. Figure 3 shows that verapamil did not cause any significant depression of the [Ca⁺⁺]_i response to LPC. The role of SL Na⁺-Ca⁺⁺ exchanger in promoting the entry of Ca⁺⁺ caused by LPC was assessed by exposing the cardiomyocytes to conditions that are known to increase Ca⁺⁺-influx through the Na⁺-Ca⁺⁺ exchange system. In fact, the reduction of extracellular Na⁺ augmented the [Ca⁺⁺]_i response to LPC significantly (fig. 4). Cardiomyocytes exposed to ouabain, an inhibitor of the sarcolemmal Na⁺-K⁺ ATPase (Vemuri et al., 1989), also showed a significant increase in both peak and plateau [Ca⁺⁺]_i upon exposure to LPC (fig. 5). The involvement of SL Na⁺-Ca⁺⁺ exchanger in the LPC-induced increase in [Ca⁺⁺]_i was examined further by use of some inhibitors of this system. Figure 6 shows that pretreatment of the cardiomyocytes with Ni⁺⁺, an inhibitor of the Na⁺-Ca⁺⁺ exchanger (O'Neill et al., 1988; Kimura et al., 1987), significantly inhibited both peak and plateau [Ca⁺⁺]_i in response to LPC. Furthermore, in cells pretreated with amiloride, another inhibitor of the exchanger (Antolini et al., 1993; Wettwer et al., 1992), the LPC-induced increase in both peak and plateau [Ca⁺⁺]_i was depressed significantly (fig. 7).

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Effect of different concentrations of LPC on [Ca++]i in adult rat cardiomyocytes. Freshly isolated adult rat cardiomyocytes were incubated with 10 µM Fura-2/AM for 40 min. The Fura-2/AM-loaded cells were exposed to varying LPC concentrations and the basal and LPC-evoked changes of peak and plateau [Ca++]i were monitored for a 5 min period by LM DMX-1100 spectrofluorometer (excitation: 340/380 nm; 510 nm). Panel A shows typical traces for different concentrations of LPC (2.5-10 µM) during a period of time. Panel B shows the bar graphs for the increase in peak [Ca++]i at different concentrations of LPC. Increase in [Ca++]i was the net increase in [Ca++]i above basal level in the absence of LPC which was 90.1 ± 6.1 nM. Values given in B are mean ± S.E. of six separate preparations.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Effect of varying concentrations of extracellular Ca++ on LPC-evoked increase in [Ca++]i. Freshly isolated adult rat cardiomyocytes were incubated with 10 µM Fura-2/AM for 40 min. The Fura-2/AM-loaded cells were exposed to 10 µM LPC for 5 min in the absence and presence of varying concentrations of extracellular Ca++. Typical traces for the LPC effect are shown in panel A. Increase in peak [Ca++]i induced by 10 µM LPC in the presence of varying concentrations of extracellular Ca++ is shown in panel B. Values given in B are mean ± S.E. of six separate preparations.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Effect of verapamil on LPC-mediated increase in [Ca++]i. Freshly isolated adult rat cardiomyocytes were incubated with Fura-2/AM for 40 min. The Fura-2/AM-loaded myocytes were incubated with varying concentrations of verapamil (10 and 20 µM) for 10 min and then exposed to 10 µM LPC for 5 min. Panel A shows the effect of LPC on [Ca++]i in the presence and absence of 20 µM verapamil; tracings before LPC addition represent the basal values. Increase in [Ca++]i caused by LPC in the presence of different concentrations of verapamil is shown in panel B. Values given in B are the peak [Ca++]i measurements and are mean ± S.E. of six preparations.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Effect of varying concentrations of extracellular Na+ on LPC-induced increase in [Ca++]i. Freshly isolated adult rat cardiomyocytes were incubated with 10 µM Fura-2/AM for 40 min. The Fura-2/AM-loaded cells were exposed to 10 µM LPC for 5 min in the presence of 42, 84 and 142 mM (control) extracellular Na+. Typical traces for the LPC effect are shown in panel A. In the experiments with 42 and 84 mM Na+, 100 mM and 58 mM choline chloride, respectively, was used to maintain the osmolarity. Increase in [Ca++]i because of LPC in the presence of different concentrations of Na+ are shown in panel B. Values given in B are the peak [Ca++]i measurements and are mean ± S.E. of six separate preparations. Plateau [Ca++]i values were 83.5 ± 4.5, 129.8 ± 9.3* and 148.3 ± 15.2* for 142 mM, 84 mM and 42 mM Na+, respectively. * represents significance (P < .05) when compared with control values (142 mM Na+).

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Stimulation of LPC-induced increase in [Ca++ ]i by ouabain. Freshly isolated adult rat cardiomyocytes were incubated with 10 µM Fura-2/AM for 40 min. The Fura-2/AM-loaded cells were incubated with varying concentrations of ouabain (1 and 2 mM) for 10 min and then exposed to 10 µM LPC for 5 min. Panel A shows the effect of LPC on [Ca++]i in the presence and absence of 2 mM ouabain. Increase in [Ca++ ]i caused by LPC at varying concentrations of ouabain is shown in panel B. Values given in B are the peak [Ca++]i measurements and are the mean ± S.E. of six preparations. Plateau [Ca++]i values were 86.6 ± 7.9, 138.8 ± 17.9* and 151.3 ± 15.7* for control, 1 mM and 2 mM ouabain, respectively. * represents significance (P < .05) when compared with control values (in the absence of ouabain).

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Inhibition of LPC-evoked increase in [Ca++]i by Ni++. Freshly isolated adult rat cardiomyocytes were incubated with 10 µM Fura-2/AM for 40 min. The Fura-2/AM-loaded cells were incubated with varying concentrations of Ni++ (2.5 and 5 mM) for 10 min and then exposed to 10 µM LPC for 5 min. Panel A shows the effect of LPC on [Ca++]i in the presence and absence of 5 mM Ni++. Increase in [Ca++]i caused by LPC at varying concentrations of Ni++ is shown in panel B. Values given in B are the peak [Ca++]i measurements and are the mean ± S.E. of six preparations. Plateau [Ca++]i values were 83.5 ± 4.5, 55.1 ± 5.3* and 47.8 ± 4.9* for control, 2.5 mM and 5 mM Ni++, respectively. * represents significance (P < .05) when compared with control values (in the absence of Ni++).

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Inhibition of LPC-mediated increase in [Ca++]i by amiloride. Freshly isolated adult rat cardiomyocytes were incubated with 10 µM Fura-2/AM for 40 min. The Fura-2/AM-loaded cells were incubated with varying concentrations of amiloride (10 and 20 µM) for 10 min and then exposed to 10 µM LPC for 5 min. Panel A shows the effect of LPC on [Ca++]i in the presence and absence of 20 µM amiloride. Increase in [Ca++]i caused by LPC at varying concentrations of amiloride is shown in panel B. Values given in B are the peak [Ca++]i measurements and are the mean ± S.E. of six preparations. Plateau [Ca++]i values were 83.5 ± 4.5, 61.5 ± 4.7* and 44.5 ± 5.5* for control, 10 and 20 µM amiloride, respectively. * represents significance (P < .05) when compared with control values (in the absence of amiloride).

Ca⁺⁺-release from SR is induced by a small amount of Ca⁺⁺ entering the cell by the Ca⁺⁺-induced Ca⁺⁺-release process (Fabiato, 1985). To assess the effect of LPC on the SR function, an inhibitor of the SR Ca⁺⁺-pump ATPase, thapsigargin (Kirby et al., 1992; Chen and Van Breemen, 1993) and a stimulator of SR Ca⁺⁺-release, ryanodine (at submicromolar concentrations) (Hansford and Lakatta, 1987; Vigne et al., 1990) were used to deplete the SR Ca⁺⁺ stores. Treatment of the cells with 20 µM thapsigargin (fig. 8) or 5 µM ryanodine (fig. 9) significantly attenuated both peak and plateau [Ca⁺⁺]_i in response to LPC.

View larger version (35K): [in this window] [in a new window]   Fig. 8.   Inhibition of LPC-evoked increase in [Ca++]i by thapsigargin. Freshly isolated adult rat cardiomyocytes were incubated with 10 µM Fura-2/AM for 40 min. The Fura-2/AM-loaded cells were incubated with varying concentrations of thapsigargin (10 and 20 µM) for 10 min and then exposed to 10 µM LPC for 5 min. Panel A shows the effect of LPC on [Ca++]i in the presence and absence of 20 µM thapsigargin. Increase in [Ca++]i caused by LPC at varying concentrations of thapsigargin is shown in panel B. Values given in B are the peak [Ca++]i measurements and are the mean ± S.E. of six preparations. Plateau [Ca++]i values were 80 ± 3.2, 85 ± 7.8 and 55.6 ± 8.2* for control, 10 µM and 20 µM thapsigargin, respectively. * represents significance (P < .05) when compared with control values (in the absence of thapsigargin).

View larger version (34K): [in this window] [in a new window]   Fig. 9.   Inhibition of LPC-mediated increase in [Ca++]i by ryanodine. Freshly isolated adult rat cardiomyocytes were incubated with 10 µM Fura-2/AM for 40 min. The Fura-2/AM-loaded cells were incubated with varying concentrations of ryanodine (1 and 5 µM) for 10 min and then exposed to 10 µM LPC for 5 min. Panel A shows the effect of LPC on [Ca++]i in the presence and absence of 5 µM ryanodine. Increase in [Ca++]i because of LPC at varying concentrations of ryanodine is shown in Panel B. Values given in B are the peak [Ca++]i measurements and are the mean ± S.E. of 6 preparations. Plateau [Ca++]i values were 80 ± 3.2, 81.6 ± 6.9 and 49 ± 4.9* for control, 1 µM and 5 µM ryanodine, respectively. * represents significance (P < .05) when compared with control values (in the absence of ryanodine).

To further assess the contribution of Na⁺-Ca⁺⁺ exchanger and SR Ca⁺⁺-regulating mechanisms in the LPC action, amiloride was used in combination with thapsigargin or ryanodine. Treatment of the cells with 10 µM amiloride plus 15 µM thapsigargin or 10 µM amiloride plus 2.5 µM ryanodine produced an additive depressant effect of these inhibitors on [Ca⁺⁺]_i (fig. 10A, B).

View larger version (25K): [in this window] [in a new window]   Fig. 10.   Inhibition of LPC-induced increase in [Ca++]i by amiloride plus thapsigargin or amiloride plus ryanodine. Freshly isolated adult rat cardiomyocytes were incubated with 10 µM Fura-2/AM for 40 min. Aliquots of the Fura-2/AM- cells were incubated with the appropriate combination of drugs for 10 min, and then exposed to 10 µM LPC for 5 min. Panel A shows the effect of LPC on [Ca++]i in the presence and absence of 10 µM amiloride plus 15 µM thapsigargin. Panel B shows the effect of LPC on [Ca++]i in the presence and absence of 10 µM amiloride plus 2.5 µM ryanodine. Tracings are representative of three separate experiments.

From the data in table 1, it can be seen that the basal [Ca⁺⁺]_i values in cardiomyocytes with several treatments under the experimental conditions used in this study were not different from those of the untreated cardiomyocytes.

	Discussion

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Although LPC is present in small quantities (0.5-3.5% of the total phospholipids) in the myocardium under physiological conditions (White, 1975), its concentration has been shown to increase because of ischemia. Such an increase in LPC concentration seems to depend on the species and tissue compartments: 120 µM in the ischemic pig myocardium (Shaikh and Downar, 1981), 197 µM in cardiac lymph and 200 µM in interstitial fluid of the ischemic region in dogs with myocardial ischemia (Akita et al., 1986), and 178 µM in the coronary sinus of humans with atrial pacing (Sedlis et al., 1990). From such observations, it appears that 100 to 200 µM concentrations of LPC may be present in the ischemic heart. However, under in vivo conditions, LPC may be bound nonspecifically to proteins in the myocardium and this may reduce the concentration of the free LPC probably by about 10 times (Corr et al., 1981; Man and Choy, 1982). Thus, 10 to 20 µM free LPC may be present in the ischemic tissue, which is compatible with the concentrations used in this study for demonstrating an increase in [Ca⁺⁺]_i in rat cardiomyocytes. Several investigators have shown the deleterious effects of LPC on myocytes within the above-mentioned pathophysiological range of concentrations (Woodley et al., 1991; Ver Donck et al., 1992; Hashizume and Abiko, 1996). Because lysophospholipids are wedge-shaped structures, their accumulation inside the membranes may alter the membrane integrity and modify the membrane protein function (Corr et al., 1984; Weltzien, 1979). Accordingly, arrhythmogenesis and contractile dysfunction caused by myocardial ischemia have been linked with LPC accumulation in the ischemic heart (Clarkson and Ten Eick, 1983; Corr et al., 1982; Pogwizd et al., 1986; Woodley et al., 1991; Ver Donck et al., 1992; Hoque et al., 1995, 1997). Liu et al. (1991) have shown that 20 µM LPC significantly increased cell shortening, produced spontaneous contractile activity and caused Ca⁺⁺ overload resulting in contracture in guinea pig ventricular myocytes. Woodley et al. (1991) detected increases in [Ca⁺⁺]_i when chick embryonic myocytes were exposed to 1 to 100 µM LPC. Thus, the results regarding the mechanisms of LPC-induced [Ca⁺⁺]_i described in the present study can be seen to address an important problem in the area of ischemic heart disease.

In this study we have shown that the increase in [Ca⁺⁺]_i in cardiomyocytes not only depends on the concentration of LPC but also is related to the concentration of Ca⁺⁺ in the medium. These observations suggest that Ca⁺⁺-influx from the extracellular space is required for the occurrence of LPC-induced increase in [Ca⁺⁺]_i in cardiomyocytes. The SL L-type channel may be responsible for the entry of extracellular Ca⁺⁺, and its activation seems to be an attractive mechanism for the LPC-induced increase in [Ca⁺⁺]_i. Five minutes exposure of myocytes to verapamil, a specific blocker of the L-type Ca⁺⁺ channel, blocked the increase in [Ca⁺⁺]_i caused by 100 µM LPC (Sedlis et al., 1983). In contrast, verapamil did not affect the LPC-induced increase in [Ca⁺⁺]_i in spontaneously contracting chick embryo cell cultures (Woodley et al., 1991) and rat cardiomyocytes (Hashizume and Abiko, 1996). Our results with verapamil are in good agreement with the latter reports demonstrating that the L-type Ca⁺⁺ channel may not be the site of action for LPC. On the other hand, Na⁺-K⁺ ATPase, which maintains the resting membrane potential and controls entry of Ca⁺⁺ indirectly via Na⁺-Ca⁺⁺ exchange, has been shown to be inhibited by LPC in SL membrane preparations from dog and rabbit hearts (Karli et al., 1979; Owens et al., 1982). However, Pitts and Okhuysen (1984) reported that the concentrations of LPC required to inhibit the Na⁺-K⁺ ATPase activity in the dog heart SL vesicles were higher than those causing changes in membrane permeability, which suggests that the inhibition of Na⁺-K⁺ ATPase by LPC may not occur to any significant extent under in vivo conditions. Our results with ouabain, a specific blocker for Na⁺-K⁺ ATPase, show an augmentation of LPC-induced increase in [Ca⁺⁺]_i in rat cardiomyocytes and support the view that Na⁺-K⁺ ATPase may be a site of action for LPC in the intact myocyte. Blockade of SL Na⁺-K⁺ ATPase would lead to an increase in the intracellular concentration of Na⁺, which in turn would bring about the reversal of the Na⁺-Ca⁺⁺ exchanger activity resulting in the entry of extracellular Ca⁺⁺ (Vemuri et al., 1989). The indirect involvement of SL Na⁺-Ca⁺⁺ exchange system also has been suggested from studies showing the LPC-induced stimulation of the Na⁺-H⁺ exchanger in intact beating rat hearts, which results in the accumulation of Na⁺ in cardiomyocytes (Hoque et al., 1997) and then produces a secondary increase in [Ca⁺⁺]_i via the Na⁺-Ca⁺⁺ exchanger (Karmazyn and Moffat, 1993).

The SL Na⁺-Ca⁺⁺ exchanger also may contribute to the LPC-induced increase in [Ca⁺⁺]_i in cardiomyocytes. This view is supported by the fact that a reduction in the extracellular concentration of Na⁺, which is known to increase Ca⁺⁺-influx, was found to augment the LPC-induced increase in [Ca⁺⁺]_i. Our results showing an attenuation of LPC-induced rise in [Ca⁺⁺]_i upon inhibition of Na⁺-Ca⁺⁺ exchanger by amiloride and Ni⁺⁺ (O'Neill et al., 1988; Kimura et al., 1987; Antolini et al., 1993; Wettwer et al., 1992), lend further support regarding the involvement of Na⁺-Ca⁺⁺ exchanger in the action of LPC. LPC also has been reported to inhibit the Na⁺-Ca⁺⁺ exchange activity in the isolated SL vesicles (Bersohn et al., 1991). The inhibition of Na⁺-Ca⁺⁺ exchange by LPC in purified SL membranes and the stimulation of Na⁺-Ca⁺⁺ exchanger in myocytes may imply that the increase in [Ca⁺⁺]_i occurs via a reduced Ca⁺⁺-efflux and an enhanced Ca⁺⁺ entry, respectively. This may be because of different sites of action of LPC on the Na⁺-Ca⁺⁺ exchanger in the SL membrane. Because Ca⁺⁺ uptake of isolated SR preparation was inhibited by 80% by 50 µM LPC, whereas the SR Ca⁺⁺-pump ATPase was depressed by about 20%, an uncoupling of Ca⁺⁺ transport and ATP hydrolytic activities has been suggested to involve SR in the mode of action of LPC (Ambudkar et al., 1988). However, LPC may not affect the SR membrane directly when exposing cardiomyocytes to this agent because LPC failed to induce an increase in [Ca⁺⁺]_i when extracellular Ca⁺⁺ was removed. On the other hand, the indirect contribution of SR in raising the [Ca⁺⁺]_i in cardiomyocytes cannot be ruled out because depletion of SR stores by inhibition of SR Ca⁺⁺-pump by thapsigargin (Kirby et al., 1992; Chen and Van Breemen, 1993) or by stimulation of SR Ca⁺⁺-release channels by low concentrations of ryanodine (Hansford and Lakatta, 1987; Vigne et al., 1990), attenuated the LPC-induced increase in [Ca⁺⁺]_i in cardiomyocytes. Thus it seems that the Ca⁺⁺-induced Ca⁺⁺-release from SR also may play an important role in eliciting the LPC-induced increase in [Ca⁺⁺]_i in cardiomyocytes. In fact, the combined blockade of Na⁺-Ca⁺⁺ exchanger and SR Ca⁺⁺-pump as well as ryanodine receptor, had an additive effect on the LPC response confirming the involvement of both mechanisms in the LPC-mediated increase in [Ca⁺⁺]_i. Accordingly, it is suggested that the LPC-induced increase in [Ca⁺⁺]_i in cardiomyocytes may be caused by its direct and indirect action on the SL Na⁺-Ca⁺⁺ exchanger for promoting the net increase in [Ca⁺⁺]_i and by an indirect action on SR for promoting the Ca⁺⁺-induced Ca⁺⁺-release.