INTRODUCTION

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THE MYOCARDIAL Na⁺/H⁺ exchange (NHE)-1 isoform is one member of a family of six found within mammalian tissue (6, 7, 11, 18, 19, 29, 36). The NHE-1 isoform is primarily located in the cardiac sarcolemmal membrane (21, 26) and functions to regulate pH through the extrusion of H⁺ in exchange for extracellular Na⁺ (6, 7, 11, 18, 19, 28, 29, 36). Of the four pH-regulating mechanisms within cardiomyocytes, the Na⁺/H⁺ exchanger is one of the most important during acidic loads (7). Others include the Na⁺/HCO₃, which functions to regulate pH at rest (7), and the Cl/HCO₃ and Cl/OH exchangers, which are more important during alkaline loads (33). The Na⁺/H⁺ exchanger is important not only during normal physiological conditions but also under pathological situations. For example, ischemia-reperfusion stimulates Na⁺/H⁺ exchange (1, 8, 10, 13-15, 24). This stimulation is thought to induce myocardial damage through cellular Ca²⁺ overload via stimulation of sarcolemmal Na⁺/Ca²⁺ exchange (8, 10, 11, 13-15, 24). Pharmacological inhibition of Na⁺/H⁺ exchange during ischemia-reperfusion has been beneficial to the myocardium (7, 10, 13-15, 24).

Despite our recognition of the importance of the Na⁺/H⁺ exchanger, our understanding of the factors that regulate the exchanger is not complete. Phosphorylation of the Na⁺/H⁺ exchange cytoplasmic domain has been identified as an important regulatory mechanism (3, 16, 30, 34, 36). Extracellular factors such as angiotensin II (9), endothelin (12), thrombin (34), and enalapril (4) also modulate Na⁺/H⁺ exchange. However, it is unclear whether the membrane in which the exchanger is embedded has any modulatory influence on the exchanger. Demaurex et al. (3) concluded that membrane phospholipids play no modulatory effect on Na⁺/H⁺ exchange (3). If true, this would be a rather unique property of the Na⁺/H⁺ exchanger. Others have reported a large alteration in the activities of ion transporters such as Na⁺/Ca²⁺ exchange, Na⁺-K⁺-ATPase, and the Ca²⁺ pump following phospholipase D (23), phosphatidylcholine-specific phospholipase C (22), phosphatidylinositol-specific phospholipase C (25), and lysophosphatidylcholine treatments (35). Since no study has directly examined the effects of membrane phospholipids on Na⁺/H⁺ exchange, this present study was carried out to evaluate the dependency of cardiac sarcolemmal Na⁺/H⁺ exchange on the phospholipid complement of the membrane.

	MATERIALS AND METHODS

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Materials. The Millipore filters, thin-layer chromatography plates, and organic solvents were supplied by Fisher Scientific. The ²²Na (0.1 ml of a 1,000 µCi/ml stock) was supplied by NEN Life Sciences, and phospholipase D (Streptomyces chromofuscus) was supplied by Calbiochem (La Jolla, CA). All other materials, including phospholipase C (Clostridium perfringens), were supplied by Sigma (St. Louis, MO).

Sarcolemmal membrane preparations. Pigs (65-85 kg) were anesthetized with Telazol (20 mg/ml) using a dose of 1 ml/23 kg animal body wt. Hearts were removed, and cardiac sarcolemmal vesicles were harvested from the left ventricle as described previously (25-27). Purity of these sarcolemmal vesicles was determined using specific marker assays. The K⁺-p-nitrophenyl phosphatase assay and the Na⁺-K⁺- ATPase assay are described elsewhere in detail (25-27). K⁺-stimulated p-nitrophenyl phosphatase activity was 12 ± 2 µmol phenol · mg¹ · h¹ in the sarcolemmal fraction (n = 7). Similarly, Na⁺-K⁺-ATPase activity in this sarcolemmal fraction was 11 ± 3 and 35 ± 10 µmol P_i · mg¹ · h¹ in the absence and presence of 12.5 µg/ml alamethicin, respectively. These activities were enriched in the sarcolemmal vesicles ~100-fold compared with homogenate. The sarcolemmal membrane-enriched final fraction was diluted into a suspension medium containing 200 mM sucrose, 25 mM MES, and 8 mM KOH, pH 5.5, and centrifuged for 2 h at 175,000 g. The pelleted membranes were resuspended in the same suspension medium at a protein concentration of 1-3 mg/ml. Protein concentrations were determined using the method described previously (25-27). These samples were frozen in liquid N₂ and stored at 80°C for subsequent analysis.

Measurement of Na⁺/H⁺ exchange. H⁺-dependent Na⁺ uptake was examined in control and phospholipase D-treated vesicles as described elsewhere in detail (26, 27). Briefly, 5 µl of ²²Na (0.1 µCi) was added to the bottom of a polystyrene tube containing 25 µl uptake medium, 200 mM sucrose, 30 mM 2-(N-hexylamino) ethanesulfonic acid (CHES), 40 mM KOH, 0.1 mM EGTA, and 0.1 mM Na⁺ (pH 10.61). A 20-µl aliquot of sarcolemmal membrane protein (11 µg) was placed on the side of the tube, and Na⁺/H⁺ exchange was initiated by vortexing the mixture. Final assay concentrations were (in mM) 180 sucrose, (in mM) 10 MES, (in mM) 17.5 CHES, (in mM) 17 KOH, (in mM) 0.05 EGTA, and (in mM) 0.05 Na⁺ at a final extravesicular pH (pH_o) of 9.33. Calibration of all assay media was done carefully using an Orion 82-10 pH electrode to ensure accuracy. After a preset time (2-5 s), 3 ml of stop solution (100 mM KCl, 20 mM HEPES, pH 7.5) was added to the polystyrene tube to arrest the reaction. The reaction mixture was filtered rapidly through 0.45-µm Millipore filters, followed by two additional 3-ml washes with the same stop solution. Filters were removed, placed in scintillation vials, and dried, and radioactivity was quantitated using scintillation spectroscopy. Blanks were treated in a similar manner except 3 ml of ice-cold stop solution was added immediately before the inclusion of 20 µl of sarcolemmal protein.

Treatment with phospholipase D. Cardiac sarcolemmal vesicles (100 µg) were exposed to phospholipase D (stock 32.5 U/ml) for 60 min at 25°C. Unless otherwise specified, the final phospholipase D-to-sarcolemmal protein ratio was 0.825 U/mg protein. Control tubes were treated in a similar manner except the enzyme was denatured in boiling water for 60 min before its use in the assay. After treatment, Na⁺/H⁺ exchange activity was examined immediately.

Phospholipid separation and quantification. Thin-layer chromatography was carried out as described previously (20, 25). After treatment with phospholipase D, phospholipids were extracted in a 2:1 chloroform:methanol mixture that contained ~5% H₂O and were separated in two dimensions on K6F silica gel thin chromatography plates. The first solvent phase was 65:25:4 chloroform:methanol:ammonium hydroxide, and the second dimension was run in 75:15:30:15:7.5 chloroform:methanol:acetone:acetic acid:H₂O to ensure complete separation of phospholipids. Acid hydrolysis using 12 N HCl was carried out between dimensions 1 and 2 to improve phosphatidic acid separation from lysophosphatidylcholine, phosphatidylinositol, and phosphatidylserine. Phospholipid spots were identified using Coomassie blue and quantified by scanning densitometry as described elsewhere (17, 32).

Statistics. Data are expressed as means ± SE. Statistical determination was done using a Student t-test and was considered significant at P < 0.05.

	RESULTS

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The effect on Na⁺/H⁺ exchange of phospholipids in the cardiac sarcolemmal membrane was determined using two phosphatidylcholine-specific phospholipases. Phospholipase D, which hydrolyzes phosphatidylcholine into phosphatidic acid, and phospholipase C, which hydrolyzes phosphatidylcholine into diacylglycerol, were used. Thin-layer chromatography was employed for both phospholipase C- and D-treated vesicles to separate the phospholipid classes. Table 1 illustrates the membrane phospholipid alterations observed following phospholipase D addition. Phospholipase D activity generated a statistically significant decrease in phosphatidylcholine in treated cardiac sarcolemmal vesicles compared with controls. A significant increase in phosphatidic acid generation following phospholipase D treatment was also observed compared with controls.

Na⁺/H⁺ exchange was examined as a function of varying concentrations of phospholipase D. There was a significant depression of Na⁺/H⁺ exchange when cardiac sarcolemmal vesicles were treated with 0.4, 0.8, and 1.6 U phospholipase D/mg sarcolemma (Fig. 1). A 30-60% decrease in ²²Na⁺ uptake was observed when measured as a function of increasing phospholipase D concentrations. This sarcolemmal Na⁺/H⁺ exchange is also sensitive to amiloride, which inhibits its activity in a dose-dependent fashion (26, 27).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   H+-dependent Na+ uptake as a function of variable concentrations of phospholipase D. Sarcolemmal vesicles were incubated with phospholipase D for 60 min at 25°C (pH 5.5). Na+/H+ exchange was examined for 5 s with a final concentration of 0.05 mM Na+, pH 9.33. Data are means ± SE of 3 separate experiments. Controls were examined in a similar manner but contained boiled, inactive phospholipase D. *P < 0.05 vs. control.

Na⁺/H⁺ exchange was measured over a number of reaction times as a function of phospholipase D treatment. Na⁺/H⁺ exchange was significantly inhibited following phospholipase D treatment at all reaction times, except at 1 min where Na⁺ uptake saturates (Fig. 2).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Time course of H+-dependent Na+ uptake in phospholipase D-treated vesicles. Sarcolemmal vesicles were incubated with 0.82 U phospholipase D/mg sarcolemma for 60 min at 25°C (pH 5.5). Na+ uptake was examined in a final solution consisting of 0.05 mM Na+, pH 9.33. Data are means ± SE of 4-7 separate experiments. *P < 0.05 vs. control.

Na⁺/H⁺ exchange was also examined at varying pH_o. In control vesicles, Na⁺ uptake exhibited an appropriate increase as the transsarcolemmal H⁺ gradient increased, as demonstrated previously (26). Phospholipase D treatment significantly inhibited Na⁺/H⁺ exchange at all pH_o values examined (Fig. 3).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   H+-dependent Na+ uptake in phospholipase D-treated sarcolemmal vesicles as a function of extravesicular pH (pHo). Sarcolemmal vesicles were preincubated with 0.82 U phospholipase D/mg protein as described. Final extravesicular Na+ concentration of the medium was 0.05 mM. Na+ uptake occurred for a period of 5 s. Data are from 4-7 separate experiments as means ± SE. *P < 0.05 vs. control.

The effect of varying the extracellular Na⁺ concentration was studied in control vesicles and vesicles treated with phospholipase D (Fig. 4). The Na⁺/H⁺ exchange is dependent on extracellular Na⁺; therefore, increasing extracellular Na⁺ should increase the amount of Na⁺ taken up into the vesicles. Na⁺/H⁺ exchange was inhibited by phospholipase D treatment at all extracellular Na⁺ concentrations examined (0.05-10 mM). These differences were statistically significant compared with controls that contained the boiled, inactive form of phospholipase D. 

View larger version (15K): [in this window] [in a new window]   Fig. 4.   H+-dependent Na+ uptake in phospholipase D-treated sarcolemmal vesicles as a function of extravesicular Na+. Sarcolemmal vesicles were incubated with 0.82 U phospholipase D/mg protein as described. The reaction time was for 5 s in a medium containing a final pH of 9.33. Data from 4-7 separate experiments are represented as means ± SE. Values are a percentage of control, where control experiments contained boiled, inactive phospholipase D. *P < 0.05 vs. control.

Although an inhibition of Na⁺/H⁺ exchange was observed, this decrease in activity may be simply due to an increase in the passive ion permeability of the sarcolemmal vesicles. Vesicles were loaded with ²²Na via Na⁺/H⁺ exchange for 1 min and then rapidly diluted into a medium that was optimal for measuring passive Na⁺ efflux from the vesicles as the Na⁺ moved down its transsarcolemmal concentration gradient (26). Consistent with the results from Fig. 2, the initial load of intravesicular Na⁺ after 1 min of intravesicular H⁺-dependent uptake was not different between control vesicles and those treated with phospholipase D (2.4 ± 0.4 and 2.1 ± 0.4 nmol/mg, respectively). Alterations in membrane fluidity and hence passive Na⁺ movement out of the vesicles may be a consequence of phospholipase D-induced exposure. Therefore, after a 1-min Na⁺ uptake time in phospholipase D-treated and untreated vesicles, passive Na⁺ efflux was initiated and measured after 2 and 15 s. This resulted in a 30 and 50% depletion of vesicular Na⁺ content. Dimethylamiloride (20 µM) was included in the efflux medium following 1 min of uptake to ensure Na⁺/H⁺ exchange was not operable during efflux. There was no statistically significant difference observed in passive Na⁺ efflux from phospholipase D-treated vesicles and control vesicles (Fig. 5).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Passive permeability of Na+ from control and phospholipase D-treated sarcolemmal vesicles. Control and treated sarcolemmal vesicles were incubated with inactive and active phospholipase D, respectively, for 60 min at 25°C in pH 5.5. After incubation, H+-dependent Na+ uptake was initiated for 1 min in a medium containing 1 mM Na+. Passive efflux was carried out in a Na+-free medium and terminated at 2 and 15 s. Data are means ± SE for 3 separate experiments.

Treatment of cardiac sarcolemmal vesicles with phospholipase D generates phosphatidic acid but also diminishes the total phosphatidylcholine pool (Table 1). To identify whether a phosphatidic acid increase or phosphatidylcholine decrease may be responsible for the change in Na⁺/H⁺ exchange, cardiac sarcolemmal vesicles were treated with a phosphatidylcholine-specific phospholipase C. Phospholipase C also depletes the phosphatidylcholine pool but generates diacylglycerol instead of phosphatidic acid. As shown in Fig. 6, Na⁺/H⁺ exchange was unaffected by phospholipase C treatment over a number of uptake times. This concentration of phospholipase C (0.05 U/mg) hydrolyzed 40% of the phosphatidylcholine content and decreased total phospholipids by 20% (n = 2). In addition, Na⁺/H⁺ exchange was examined over several phospholipase C concentrations (0.01, 0.03, and 0.05 U/mg). There was no statistically significant difference in Na⁺/H⁺ exchange in phospholipase C-treated vesicles compared with controls at any of the phospholipase C concentrations studied (P < 0.05). The activity was 102 ± 8, 112 ± 11, and 93 ± 10 (as a percentage of control activity) in vesicles treated with 0.01, 0.03, and 0.05 U/mg phospholipase C, respectively (n = 3).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   H+-dependent Na+ uptake in phospholipase C-treated sarcolemmal vesicles as a function of reaction time. Sarcolemmal vesicles were incubated with 0.05 U phospholipase C/mg protein for 60 min at 25°C in pH 5.5. After treatment, Na+ uptake occurred for 2 s to 10 min in a final medium consisting of 0.05 mM Na+, pH 9.33. Controls were examined in similar manner but contained boiled, inactive phospholipase C. Data are means ± SE of 3-4 separate experiments. Data are presented in phospholipase C-treated vesicles as a percentage of control, untreated vesicles.

	DISCUSSION

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This study demonstrates that the cardiac sarcolemmal Na⁺/H⁺ exchanger requires a suitable phospholipid environment for optimal activity and that phospholipase D-induced changes in the exchanger-associated phospholipids inhibit the exchanger activity. After phospholipase D treatment, all parameters of Na⁺/H⁺ exchange (Na⁺, pH_o, and reaction time dependency) were inhibited. The striking difference between the effects of phospholipase C and D on Na⁺/H⁺ exchange strongly implicates phosphatidic acid as the lipid moiety responsible for the effects on Na⁺/H⁺ exchange. Changes in the membrane phosphatidylcholine content by up to 40% with phospholipase C treatment did not alter Na⁺/H⁺ exchange. However, phospholipase D inhibited Na⁺/H⁺ exchange by 30-60% compared with controls. Phospholipase D hydrolyzes phosphatidylcholine as well but instead generates phosphatidic acid. However, without further in-depth studies, it is not possible to identify the precise phospholipid class responsible for the effects on Na⁺/H⁺ exchange. It is also possible that phospholipase D action may inhibit Na⁺/H⁺ exchange through an alteration of the phosphatidylcholine-to-phosphatidic acid ratio (5). An increase in passive Na⁺ permeability may also account for the observed inhibition. However, when passive efflux was examined, no difference between control and phospholipase D-treated cardiac sarcolemmal vesicles was observed. Alternatively, a decrease in the total volume of the vesicles may also account for the observed inhibition. However, when H⁺-dependent Na⁺ uptake was examined as a function of time, there was no difference between control and phospholipase D-treated vesicles following 1 min of Na⁺ uptake. Since total intravesicular Na⁺ loading capacity was unaltered, this suggests vesicular size and volume were not changed in a meaningful manner.

In contrast to the conclusions of others (3), our data argue persuasively that membrane phospholipids can modulate Na⁺/H⁺ exchange activity. The previous work did not directly examine the effects of endogenous phospholipids on Na⁺/H⁺ exchange (3). Instead, the conclusions were based on effects observed where phospholipid flippase activity was stimulated. The flippase allows for preferential distribution of negatively charged phospholipids to the inner leaflet of the phospholipid bilayer (37). The differences in methodology are likely critical for contrasting results and conclusions. Our data provide evidence for the regulatory role of membrane phospholipids on Na⁺/H⁺ exchange, which the previous paper does not. Our results demonstrating a lack of effect of changes in membrane phosphatidylcholine content on Na⁺/H⁺ exchange are, therefore, consistent with the conclusions of Demaurex et al. (3). However, the present results extend these observations to suggest class-dependent effects of specific phospholipids on the exchanger. Such effects are not unprecedented. The degree of inhibition generated by phospholipase D treatment in the present study is also similar to that observed previously by Na⁺-K⁺-ATPase and Ca²⁺-ATPase (23). The Na⁺/Ca²⁺ exchanger also exhibits a phospholipid-specific modulatory response (25).

In summary, our results demonstrate an effect of phospholipase D on exchange activity. It will be of interest in the future to examine whether the alterations in phospholipase D activity observed in postischemic reperfused hearts (2) and during ischemic preconditioning (31) may influence cardiac injury via an effect on Na⁺/H⁺ exchange.