INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

LYSOPHOSPHATIDYLCHOLINE (LPC) is one of the products of breakdown of membrane phospholipids, formed from phosphatidylcholine by hydrolysis of the sn-2 fatty acid by phospholipase A₂ (PLA₂). The accumulation of LPC may contribute to arrhythmias and mechanical dysfunction of the heart induced by ischemia and reperfusion (4, 10). It has been reported that exogenous LPC increases the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the normal cardiac cells (17) and inflicts injury on the heart cell similar to that induced by ischemia and reperfusion (10). Previous studies have demonstrated that the LPC-induced increase in [Ca²⁺]_i is accompanied by hypercontracture of cells and release of cytosolic enzymes from the cell (1, 23), indicating that LPC inflicts irreversible damage on the cell. However, the mechanisms of the LPC-induced cell damage remain unclear.

We have previously shown that LPC inflicts injury on the cell without an increase in [Ca²⁺]_i under Ca²⁺-free conditions (2). However, in the presence of Ca²⁺, LPC produces an increase in [Ca²⁺]_i, and the Ca²⁺ overload is the prominent event that directly leads to cell death. It is therefore important to clarify the mechanisms of the cell damage induced by LPC under the Ca²⁺-present conditions. Because LPC produces an increase in [Ca²⁺]_i that may activate PLA₂, it is possible that the cell damage induced by exogenous LPC is related to an enhanced PLA₂ activity that degrades membrane phospholipids. Moreover, the newly produced metabolites, such as fatty acids, and new lysophosphatides, including LPC, liberated from membrane phospholipids by the activation of PLA₂, may accelerate the damage of the cell, thereby forming a vicious cycle. If this is true, inhibition of the PLA₂ activity would block the cycle and provide a protective effect against the cell damage induced by LPC. To test this hypothesis, the effects of three PLA₂ inhibitors, 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid (DEDA), 3-(4-octadecylbenzoyl)acrylic acid (OBAA), and manoalide, on the LPC-induced changes in [Ca²⁺]_i, cell shape, release of creatine kinase (CK), and level of nonesterified fatty acids (NEFA) were determined in isolated rat cardiomyocytes.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Isolation of myocytes. Ca²⁺-tolerant cardiomyocytes were isolated from male Sprague-Dawley rats (~250 g body wt). The isolation procedure has been described in detail in a previous study (8). Briefly, the heart was quickly removed and perfused with a Langendorff perfusion apparatus for 3 min with Ca²⁺-free Krebs-Ringer bicarbonate (KRB) solution and then perfused for another 30 min with 25 µM CaCl₂, 0.1% collagenase, and 0.1% BSA. Thereafter, ventricular tissue was chopped and incubated for 10 min in the same medium supplemented with 1% BSA. The tissue suspension was gently agitated to release the myocytes and filtered through three-layer gauze to remove the connecting tissue. The resultant myocytes were washed twice with fresh KRB buffer containing 1% BSA and 25 µM CaCl₂, and then the Ca²⁺ concentration was increased to 1.0 mM gradually. Finally, the cardiomyocytes were suspended in KRB buffer containing (in mM) 119 NaCl, 15 NaHCO₃, 2.6 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 11 glucose, and 1.0 CaCl₂, bubbled with 5% CO₂-95% O₂. In the Ca²⁺-free study, myocytes were suspended in KRB buffer in which CaCl₂ was omitted and 0.5 mM EGTA was added. All experiments were performed at 37°C.

Measurement of [Ca²⁺]_i. The suspension of myocytes was diluted to a concentration of 5% (vol/vol) (e.g., 0.5 ml pellet in 10 ml of KRB buffer) with the KRB buffer and then incubated with 5 µM fura 2-acetoxymethyl ester (fura 2-AM). The cell suspension (in the KRB buffer) was constantly bubbled with 5% CO₂-95% O₂ at room temperature for 1 h. Thereafter, 1 ml of cell suspension was taken and washed twice with fresh KRB buffer before use. The fluorescence intensities were measured by using a fluorescence spectrometer (model CAF-110; Japan Spectroscopic, Tokyo, Japan). The excitation wavelengths were 340 and 380 nm, and the emission wavelength was 510 nm. Calibration of the fluorescence intensity of fura 2 was done at the end of each experiment with the addition of digitonin (100 µM) and EGTA (20 mM) to the cell suspension to obtain the maximum and minimum fluorescence levels, respectively. The [Ca²⁺]_i was estimated according to the method described by Grynkiewicz et al. (7).

Observation of morphological change. The myocyte samples were fixed in 2.5% glutaraldehyde. About 150 myocytes were counted under a light microscope and subjected to study. The number of rod-shaped cells expressed as a percentage of the total number of cells was used as an indicator of the morphological change.

Measurement of CK. The CK activity was measured by colorimetric method with a kit purchased from Sigma Chemical (kit no. 520-C; St. Louis, MO). The cell samples were centrifuged, and CK was extracted from pellets (cells) with 1% Triton X-100. The CK activity in both the supernatant and the cellular extract was measured. The activity of CK released into the supernatant was expressed as a percentage of the total CK activity (supernatant plus pellets).

Determination of NEFA in myocytes. The determination of NEFA in myocytes was performed according to the method described in our previous study (12). Briefly, the NEFA were extracted from the cell suspension with chloroform-methanol (2:1) containing 0.05% butylated hydroxytoluene as an antioxidant. The extracted NEFA were converted into their fluorescent derivatives of 9-anthryldiazomethane (ADAM). The resultant mixture was kept at room temperature in a dark room for 3 h and then filtered through a Millipore filter (FH 0.5 µm; Nihon Millipore Kogyo K. K., Yonezawa, Japan). The ADAM derivatives of NEFA were separated by reversed-phase HPLC with a Zorbax-ODS column (0.46 × 25 cm; Du Pont, Philadelphia, PA) using methanol-water (2,000:180, vol/vol) as a mobile phase. The column temperature was maintained at 60°C, and the flow rate of the mobile phase was set at 1.2 ml/min. The quantitative analysis was performed by comparing the peak height of fluorescence intensity with that of a known amount of heptadecanoic acid (C_17:0). The amount of NEFA was expressed as picomoles per milligram of protein. The protein in the myocyte was measured by the microbiuret method.

Materials. LPC (L--lysophosphatidylcholine, palmitoyl), DEDA, OBAA, and manoalide were purchased from Sigma Chemical (St. Louis, MO). Fura 2-AM was purchased from Dojindo Laboratory (Kumamoto, Japan). DEDA and manoalide were dissolved in 99.5% ethanol, and OBAA was dissolved in DMSO, and they were further diluted with distilled water. The final concentration of ethanol or DMSO was <0.08%, which did not affect [Ca²⁺]_i and cell shape. All the solutions were prepared immediately before use. The IC₅₀ values of manoalide, DEDA, and OBAA were estimated to be 13, 16, and 0.07 µM, respectively, based on their inhibitory effects on PLA₂ activity examined in the P388D₁ cell or porcine pancreas (14, 15).

Statistical analysis. Results are given as means ± SE. In all experiments, n indicates the number of experiments. Significance of difference was determined with an ANOVA followed by Dunnett's multiple comparisons test with a significance level of P < 0.05.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Effects of LPC on [Ca²⁺]_i, cell shape, and release of CK in presence or absence of Ca²⁺. In the normal KRB buffer containing 1 mM Ca²⁺, addition of LPC (15 µM) to the myocytes produced a rapid and pronounced increase in [Ca²⁺]_i, hypercontracture of the cells, resulting in a decrease in the number of rod-shaped cells, and an increase in the release of CK. As shown in Fig. 1, 5 min after the addition of LPC, [Ca²⁺]_i increased from ~80 to ~2,500 nM, the number of rod-shaped cells expressed as a percentage of the total number of cells decreased from ~70 to ~5%, and the amount of released CK expressed as a percentage of the total amount of CK significantly increased from 11.4 ± 2.1 to 69.7 ± 2.8%. The effects of LPC on [Ca²⁺]_i, cell shape, and release of CK were further examined in the absence of extracellular Ca²⁺ by the application of a Ca²⁺-free KRB buffer, in which Ca²⁺ was omitted and 0.5 mM EGTA was added. Under the Ca²⁺-free conditions, LPC did not increase [Ca²⁺]_i, but it changed the cell shape and increased the release of CK to a degree similar to that in the presence of Ca²⁺ (Fig. 1), suggesting that Ca²⁺ overload is not a cause of the LPC-induced cell membrane damage.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Effects of lysophosphatidylcholine (LPC) on intracellular Ca2+ concentration ([Ca2+]i), cell shape, and release of creatine kinase (CK) in normal Krebs-Ringer bicarbonate (KRB) (n = 5-8) or Ca2+-free buffer (n = 5) in isolated rat cardiomyocytes. In Ca2+-free buffer, CaCl2 was omitted from normal KRB buffer to which 0.5 mM EGTA was added. Data were obtained before and 5 min after addition of LPC. Number of rod-shaped cells is expressed as a percentage of total number of cells. Activity of CK released into supernatant solution is expressed as a percentage of total CK activity. ** P < 0.01 compared with corresponding value before LPC.

Change in NEFA levels induced by LPC and effects of phospholipase A₂ inhibitors. The levels of individual NEFA, including saturated (C_14:0, myristic acid; C_18:0, stearic acid) and unsaturated NEFA (C_16:1, palmitoleic acid; C_18:1, oleic acid; C_18:2, linoleic acid; C_20:4, arachidonic acid), in the myocytes were measured in the presence or absence of Ca²⁺. The results are shown in Figs. 2 and 3. In the normal KRB buffer containing 1 mM Ca²⁺, exposure of the myocytes to LPC for 5 min did not alter the level of C_14:0, but it increased the level of other NEFA, including C_18:0, C_16:1, C_18:1, C_18:2, and C_20:4, significantly. Levels of the four unsaturated NEFA (C_16:1, C_18:1, C_18:2, and C_20:4) increased about three- to fourfold after the application of LPC. In the Ca²⁺-free buffer containing 0.5 mM EGTA, LPC induced significantly less accumulation of unsaturated NEFA than in the presence of Ca²⁺. In the presence of Ca²⁺, preincubation of the myocytes with DEDA, OBAA, or manoalide at the concentration of 20 µM partially but significantly attenuated the LPC-induced accumulation of the four unsaturated NEFA to a similar degree (P < 0.05), and the levels of NEFA that have been suppressed by the PLA₂ inhibitors were at approximately the same levels as those in the absence of Ca²⁺. DEDA, OBAA, or manoalide had little effect on the LPC-induced accumulation of C_18:0.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Increase in levels of saturated nonesterified fatty acids (NEFA; C14:0 and C18:0) induced by LPC in normal KRB buffer and effects of 3 phospholipase A2 (PLA2) inhibitors [7,7-dimethyl-(5Z,8Z)-eicosadienoic acid (DEDA), 3-(4-octadecylbenzoyl)acrylic acid (OBAA), and manoalide] on LPC-induced increase in levels of saturated NEFA in isolated rat cardiomyocytes. Cells were preincubated with respective inhibitors for 5 min and then exposed to LPC for 5 min. Each value represents a mean ± SE (n = 5). # P < 0.05 compared with values in group with no treatment.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Increase in levels of unsaturated NEFA (C16:0, C18:1, C18:2, and C20:4) induced by LPC in normal KRB buffer and effects of 3 PLA2 inhibitors (DEDA, OBAA, and manoalide) on LPC-induced increase in levels of unsaturated NEFA in isolated rat cardiomyocytes. Cells were preincubated with respective inhibitors for 5 min and then exposed to LPC for 5 min. Each value represents a mean ± SE (n = 5). *P < 0.05, **P < 0.01 compared with values in LPC group. # P < 0.05, ## P < 0.01 compared with values in group with no treatment.

Effects of phospholipase A₂ inhibitors on LPCinduced increase in [Ca²⁺]_i. The effects of three PLA₂ inhibitors (DEDA, OBAA, and manoalide) on the LPC-induced increase in [Ca²⁺]_i in the presence of Ca²⁺ were examined (Fig. 4). The myocytes were preincubated with each of the inhibitors at a concentration of 10, 20, or 50 µM for 5 min, and then LPC (15 µM) was added to the myocytes. The [Ca²⁺]_i values 5 min after the addition of LPC were used to evaluate the effects of the three compounds. DEDA and OBAA at a concentration of 20 or 50 µM significantly attenuated the LPC-induced increase in [Ca²⁺]_i (P < 0.05), whereas manoalide (20 or 50 µM) did not.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effects of DEDA, OBAA, and manoalide on LPC-induced increase in [Ca2+]i in isolated rat cardiomyocytes. Cells were preincubated with respective inhibitors for 5 min, and then LPC was added. Data were obtained before and 5 min after addition of LPC. Each value represents a mean ± SE (n = 5-8). Nos. in columns are concentrations (in µM) of respective inhibitors. * P < 0.05 compared with values in respective LPC group.

Effects of phospholipase A₂ inhibitors on LPCinduced change in cell shape in presence of Ca²⁺. The samples for the observation of morphological change were those used for the measurements of [Ca²⁺]_i, which were taken 5 min after the addition of LPC. Five minutes after the addition of LPC, the number of rod-shaped cells as a percentage of the total number of cells decreased from ~70 to ~5%. Consistent with the [Ca²⁺]_i data, DEDA or OBAA at a concentration of 20 or 50 µM significantly attenuated the change in cell shape induced by LPC (P < 0.01), whereas manoalide (20 or 50 µM) did not (Fig. 5).

View larger version (43K): [in this window] [in a new window]   Fig. 5.   Effects of DEDA, OBAA, and manoalide on LPC-induced decrease in number of rod-shaped cells expressed as a percentage of total cells in isolated rat cardiomyocytes. Samples are those described in Fig. 4. Data were obtained before and 5 min after addition of LPC. Each value represents a mean ± SE (n = 5-8). Nos. in columns are concentrations (in µM) of respective inhibitors. ** P < 0.01 compared with values in respective LPC group.

Effects of phospholipase A₂ inhibitors on the LPC-induced release of CK in presence of Ca²⁺. The amount of released CK expressed as a percentage of the total amount of CK was 11.4 ± 2.1% in the cells 5 min after incubation without LPC. The amount of released CK significantly increased to 69.7 ± 2.8% after the addition of LPC (15 µM) for 5 min. Both DEDA and OBAA at a concentration of 20 µM significantly attenuated the release of CK induced by LPC (P < 0.05), whereas manoalide (20 µM) did not (Fig. 6).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Effects of DEDA, OBAA, and manoalide on the LPC-induced release of CK in isolated rat cardiomyocytes. Cells were preincubated with respective inhibitors for 5 min and exposed to LPC for 5 min. Each value represents a mean ± SE (n = 6). *P < 0.05 compared with values in LPC group.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

It has been reported that the concentration of LPC increases in both tissue and extracellular fluid during ischemia. Shaikh and Downar (19) reported that the level of LPC measured in ischemic myocardium increased from ~14 to 23 µM, and Snyder et al. (20) reported that the concentration of LPC in venous effluent from ischemic myocardium increased from ~100 to 200 µM. Exogenous LPC, at a concentration of 10 µM, has been shown to produce cardiac injury similar to that induced by ischemia (10). In cardiomyocytes, LPC at concentrations ranging from 3 to 80 µM produces Ca²⁺ overload (17, 23). In the present study we used 15 µM LPC, which exerted a marked increase in [Ca²⁺]_i, morphological change, and an increase in the release of CK.

The aim of this study was to examine whether LPC activates PLA₂ and whether the activation of PLA₂ is responsible for the LPC-induced cell damage in terms of change in cell shape and release of CK. In the present study LPC produced a rapid increase in the [Ca²⁺]_i, a change in cell shape from rod to round, and an increase in the release of CK accompanied by the accumulation of NEFA, predominantly the unsaturated NEFA, including arachidonic acid. Because unsaturated NEFA are usually incorporated to glycerol backbound at the sn-2 position by an acyl bond that is specifically hydrolyzed by PLA₂, the fact that there is accumulation of unsaturated NEFA, especially arachidonic acid, which preferentially incorporates to the sn-2 position, suggests that there is an increase in PLA₂ activity. These results clearly indicate that LPC increases the PLA₂ activity.

Degradation of membrane phospholipids by the activation of PLA₂ is believed to play a role in the loss of membrane integrity (3, 18). If the cell damage induced by LPC is caused by the enhanced PLA₂ activity, inhibition of PLA₂ activity should provide a protective effect against the cell damage induced by LPC. In the heart, three types of PLA₂ are found. The first type is the membrane-associated, low-molecular-weight PLA₂, which requires a millimolar concentration of Ca²⁺. The second type is the cytosolic PLA₂, which requires a submillimolar concentration of Ca²⁺, and the third type is the plasmalogen-selective PLA₂, which is Ca²⁺ independent (21). DEDA, OBAA, and manoalide have been reported to inactivate Ca²⁺-dependent PLA₂ activity purified from several sources, such as cobra or bee venom and porcine pancreas. In addition, it was reported that DEDA and manoalide inhibit the Ca²⁺-dependent membrane-associated phospholipase A₂ (15). In the present study, DEDA, OBAA, and manoalide significantly attenuated the accumulation of unsaturated NEFA induced by LPC, indicating that these three compounds also inhibit Ca²⁺-dependent PLA₂ activity of cardiomyocytes. Unexpectedly, however, it was found that DEDA, OBAA, and manoalide, which inhibited the LPC-induced increase in PLA₂ activity to a similar degree, exerted different effects on the changes induced by LPC. DEDA and OBAA attenuated the LPC-induced increase in [Ca²⁺]_i, change in cell shape, and release of CK. Manoalide, however, failed to attenuate the LPC-induced increase in [Ca²⁺]_i, change in cell shape, and release of CK. It has been reported that manoalide could also inhibit phospholipase C, resulting in inhibition of the [Ca²⁺]_i increase mediated by inositol 1,4,5-trisphosphate and 1,2-diacylglycerol (5). This effect, however, could not be considered to diminish the beneficial effects of manoalide on the LPC-induced cell damage. Thus the differential effects of three PLA₂ inhibitors on the LPC-induced cell damage suggest that activation of the Ca²⁺-dependent PLA₂ may not be a primary cause of the cell damage induced by LPC.

The foregoing view was further confirmed by the study under Ca²⁺-free conditions. In the absence of Ca²⁺, LPC did not increase [Ca²⁺]_i and produced significantly less accumulation of NEFA than resulted from the Ca²⁺-activated PLA₂, but it still changed the cell shape and increased the release of CK to a degree similar to that released in the presence of Ca²⁺. These results strongly suggest that activation of PLA₂ is a secondary event rather than a primary cause of the LPC-induced cell damage. Having an amphiphilic property, LPC associates with the membrane (16) to produce the deleterious effects on the cell membrane, such as change in membrane fluidity (6) and an increase in membrane permeability (13). It is therefore reasonable to assume that LPC directly inflicts damage on the cell membrane, resulting in an increase in release of enzymes from the cytosol, and, hence, increases [Ca²⁺]_i and activates PLA₂, thereby elevating the level of NEFA. Accordingly, it is suggested that activation of PLA₂ may not play a primary role in the initiation of the cell damage induced by LPC.

Recently, Hoque et al. (11) reported that Na⁺/H⁺ exchange inhibitors protect against cardiac injury induced by LPC, with very effective protection against 3 µM LPC, reduced benefit against 5 µM LPC, and total lack of protection against 10 µM LPC, indicating that different concentrations of LPC may produce damage by different mechanisms. It is therefore unclear whether PLA₂ plays a role in the cell damage induced by lower concentrations of LPC. In addition, the plasmalogen-selective PLA₂, which does not require Ca²⁺ for its activation, has been identified in heart tissue (24). Hazen and Gross (9) reported that predominant PLA₂ activity in the myocardium is Ca²⁺ independent. DEDA, OBAA, and manoalide are all Ca²⁺-dependent PLA₂ inhibitors (14, 15), and therefore it is possible that the Ca²⁺-independent PLA₂ is also activated by the addition of LPC. Further study is needed to determine the extent to which the Ca²⁺-independent PLA₂ participates in the initiation of cell damage induced by LPC. DEDA and OBAA, which attenuated the LPC-induced cell damage, may have actions other than the PLA₂ inhibitory effect that are responsible for protection of the LPC-induced cell damage.

In summary, the results demonstrate that LPC produces an increase in [Ca²⁺]_i, a change in cell shape, a release of CK, and an accumulation of unsaturated NEFA, including arachidonic acid, indicating that LPC induces cell damage accompanied by activation of PLA₂. The three PLA₂ inhibitors, DEDA, OBAA, and manoalide, attenuated the NEFA accumulation to a similar degree; however, only DEDA and OBAA attenuated the increase in [Ca²⁺]_i, the change in cell shape, and the release of CK, whereas manoalide did not. In the absence of Ca²⁺, LPC did not increase [Ca²⁺]_i with significantly less accumulation of NEFA, but LPC still changed the cell shape and increased the release of CK to a degree similar to that released in the presence of Ca²⁺. These results suggest that the enhanced activity of PLA₂ may not mainly contribute to the cell damage induced by LPC.