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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1561-1567.)
© 1997 American Heart Association, Inc.

Articles

Lysophosphatidylcholine Inhibits Receptor-Mediated Ca²⁺ Mobilization in Intact Endothelial Cells of Rabbit Aorta

Yoichi Miwa; Ken-ichi Hirata; Seinosuke Kawashima; Hozuka Akita; ; Mitsuhiro Yokoyama

From the First Department of Internal Medicine, Kobe University School of Medicine, Japan.

Correspondence to Mitsuhiro Yokoyama, MD, First Department of Internal Medicine, Kobe University School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650, Japan.

	Abstract

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Abstract We have previously reported that lysophosphatidylcholine (LPC), which accumulates in oxidized LDL and atherosclerotic arteries, inhibits endothelium-dependent relaxation and modulates Ca²⁺ regulation in cultured bovine aortic endothelial cells. To test the effect of LPC on endothelium-dependent relaxation and endothelial Ca²⁺ regulation in intact vessels, we simultaneously measured both isometric tension and endothelial cytosolic free Ca²⁺ concentration ([Ca²⁺]_i), using fura 2, in intact endothelial cells of aortic strips isolated from rabbits. In the aortic strips precontracted with phenylephrine, cumulative addition of acetylcholine (ACh) dose dependently induced endothelium-dependent relaxation, with an increase in endothelial [Ca²⁺]_i, and positive correlation was obtained between these two parameters. LPC (2 to 20 µmol/L) inhibited both ACh (3 µmol/L)-induced endothelium-dependent relaxation and an increase in endothelial [Ca²⁺]_i in a dose-dependent manner. On the other hand, phosphatidylcholine (20 µmol/L) affected neither ACh-induced endothelium-dependent relaxation nor an increase in endothelial [Ca²⁺]_i. LPC had no effect on endothelium-independent relaxation and a decrease in smooth muscle [Ca²⁺]_i induced by nitroglycerin. Thus, the inhibitory effect of LPC on endothelium-dependent relaxation is due to the inhibition of agonist-induced Ca²⁺ mobilization in vascular endothelial cells, which is an essential step in the synthesis of endothelium-derived relaxing factor.

Key Words: phospholipids • vascular endothelium • lysophosphatidylcholine • endothelium-derived relaxing factor • intracellular calcium

	Introduction

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Endothelium-dependent relaxation is markedly reduced in atherosclerotic arteries obtained from experimental animals and humans,^{1 2 3 4 5} and this impaired endothelium-dependent relaxation in atherosclerosis is thought to play an important role in the pathogenesis of ischemic heart disease.^{6 7} Ox-LDL has been implicated as an important cause of atherosclerosis, and its presence in atherosclerotic lesions of rabbits and humans has been demonstrated.⁸ One of the properties of ox-LDL is its increased LPC content. LPC concentration is increased manyfold in atherosclerotic arteries.⁹ We^{10 11} and another group¹² have previously demonstrated that ox-LDL inhibits endothelium-dependent relaxation of rabbit aorta and that LPC generated during oxidative modification of LDL is the main factor in the impairment of endothelium-dependent relaxation.

In endothelial cells, it is generally accepted that a rise in [Ca²⁺]_i is an essential step in the synthesis of EDRF.^{13 14 15 16} Numerous studies have shown that the agonist-induced increase in [Ca²⁺]_i involves both a transient inositol 1,4,5-triphosphate–mediated release of Ca²⁺ from intracellular stores and a more sustained transmembrane influx of Ca²⁺ from the extracellular space. We have already reported that LPC inhibits bradykinin-induced phosphoinositide hydrolysis and Ca²⁺ transients in cultured bovine aortic endothelial cells.¹⁷ The limitations of the study are that the cultured endothelial cells may exhibit phenotypic modifications, including receptor and ion channel expression, an intracellular signal-transduction system, and their functions, compared with intact endothelial cells. Furthermore, using intact endothelial cells isolated from rabbits whose physiological processes (eg, intercellular communication) have not been altered by cell isolation and culture cell passages, we simultaneously measured both isometric tension and [Ca²⁺]_i in the intact aortic endothelial cells to investigate the effect of LPC on the receptor agonist–evoked rise in endothelial [Ca²⁺]_i in the present study.

	Methods

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Simultaneous Measurement of Isometric Tension and [Ca²⁺]_i in Aortic Strips
Japanese white rabbits (2.5 to 3.5 kg) were anesthetized with pentobarbital sodium (30 mg/kg body weight IV), and the descending thoracic aorta was isolated and cleaned of surrounding tissue. Aortic rings 2 mm wide were cut and opened. Two types of muscle strips were prepared: (1) strips with endothelium and (2) strips in which endothelium was mechanically removed by rubbing the intimal surface with a filter paper moistened with PSS of the following composition (in mmol/L): NaCl 140, KCl 5.4, CaCl₂ 1.5, MgCl₂ 1.0, NaHCO₃ 23.8, EDTA 0.01, and glucose 5.5, saturated with 95% O₂ and 5% CO₂, at 37°C and pH 7.4. High-K⁺ solution was made by substituting NaCl with equimolar KCl.

Isometric tension and [Ca²⁺]_i in aortic strips were measured simultaneously as previously described.^{18 19} The aortic strips were treated with 6 µmol/L fura 2–AM in the presence of 0.02% cremophor EL, a noncytotoxic detergent, for 4 to 6 hours at room temperature (20°C to 25°C) under protection from light. The aortic strip was held horizontally on a silicon rubber sheet laid on the quartz bottom of a 5-mL-vol organ bath (37°C) attached to a fluorimeter (CAF-110, Japan Spectroscopic). One end of the strip was pinned to the silicon rubber sheet adventitial side up and another end was connected to a strain-gauge transducer (Orientec) to monitor the isometric tension under a resting tension of 1 g. The fura 2–loaded muscle strips were rinsed twice with normal PSS for 15 minutes in the bath. Excitation light (a spot 2 to 3 mm in diameter) was focused on the strip through a slit of the sheet from the bottom of the bath. The light was obtained from a xenon high-pressure lamp (75 W) equipped with a rotating wheel that had 340-nm and 380-nm interference filters. The aortic strip was illuminated alternately at a cycle of 48 Hz with two excitation wavelengths (340 and 380 nm), and fluorescence emitted from the strip was collected into a photomultiplier through a 500-nm filter. The intensity of fluorescence induced by excitation at 340 nm (F340) and that induced by excitation at 380 nm (F380) was measured, and the ratio of these two fluorescence values (F340/F380) was calculated automatically. Fura 2–Ca²⁺ fluorescence was detected from the endothelial surface of the strip with endothelium and from the surface of the smooth muscle layer of the strip without endothelium.

After muscle tension and fluorescence (F340, F380, and F340/F380) stabilized, the tissue was conditioned by application of high K⁺ (72.7 mmol/L). Then the strips were precontracted with 0.3 µmol/L PE and subsequently relaxed by a cumulative addition of ACh. After washout and equilibration, the strips were preincubated with selected concentrations of PC or LPC for 20 minutes and the contraction-relaxation cycle was repeated. In some experiments, effects of phospholipids on NTG-induced response were examined in the strips with or without endothelium.

In the fura 2–unloaded strips, we examined the changes in autofluorescence in a preliminary experiment and confirmed that muscle contraction increased both F340 and F380 and that inasmuch as the increments in these lights were proportional, F340/F380 did not change. Similar results were reported in rat aortic strips.²⁰ Therefore, the influence of autofluorescence on the changes in [Ca²⁺]_i was negligible in the present study. In the aortic strips successfully loaded with fura 2, an increase of [Ca²⁺]_i induced an increase in F340, as well as a decrease in F380, and resulted in an increase in F340/F380. On the other hand, the insufficient fura 2 loading or the movement of the muscle strip did not change F340 and F380 in mirror image. To distinguish the fura 2–Ca²⁺ signal from autofluorescence or movement artifact, F340 and F380 were always monitored. Only those preparations in which F340 and F380 changed in mirror image were used in the present study.

We measured muscle tension and F340/F380 at a level of their sustained phase in each dose of relaxant agents. The absolute [Ca²⁺]_i was calculated from the equation [Ca²⁺]_i=K_dx(R-R_min)/(R_max -R)xs.²¹ K_d, the dissociation constant of the Ca²⁺–fura 2 complex, was assumed to be 224 nmol/L; R represents the experimentally determined F340/F380. R_max and R_min were measured in the presence of 2 µmol/L bromo-A23187, the nonfluorescent Ca²⁺ ionophore, and 5 mmol/L EGTA, respectively. The constant value s is the ratio of F380 of the tissue measured in Ca²⁺-free solution to that measured in Ca²⁺-containing solution in the presence of bromo-A23187. Relaxation values and NTG-induced [Ca²⁺]_i were expressed as percent decreases of the PE (0.3 µmol/L)-induced response. ACh (3 µmol/L)-induced [Ca²⁺]_i was expressed as percent values of that before treatment with phospholipids.

Data Analysis and Statistics
Data were expressed as mean±SEM. Comparisons of means were made by using the Student's t test for unpaired samples; when more than two means were compared, ANOVA and the Bonferroni test for samples were used. A value of P<.05 was considered statistically significant.

Drugs
The following drugs were used: 1-palmitoyl-2-oleoyl-PC, 1-palmitoyl-LPC, 1-PE hydrochloride, ACh chloride, substance P, and bromo-A23187 (Sigma Chemical Company); NTG (Nihonkayaku); fura 2–AM and EGTA (Dojindo Laboratories); and cremophor EL (Nakarai Chemicals). PC and LPC, stored in chloroform-methanol mixtures at -20°C, were dried under a stream of N₂ gas, dissolved in distilled water, and then sonicated just before use. Bromo-A23187 was dissolved in ethanol to make the stock solution. Maximal ethanol concentration in the organ bath was 0.02%, which did not induce any changes in vascular tension and [Ca²⁺]_i. The other drugs were dissolved in distilled water and then diluted in buffer. All were expressed as final concentrations.

	Results

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Effect of ACh and NTG in PE-Stimulated Aorta With or Without Endothelium
Typical recordings of the responses to ACh and NTG in aortic strips with or without endothelium are shown in Fig 1. Cumulative addition of ACh dose dependently induced increases in [Ca²⁺]_i and relaxations in aortic strips with endothelium (Fig 1A, top). In the presence of endothelium, [Ca²⁺]_i levels in the resting state were 115±7 nmol/L, and ACh (3 µmol/L) increased [Ca²⁺]_i to 544±82 nmol/L. As shown in Fig 2, it was found that there is a positive correlation between endothelial [Ca²⁺]_i and relaxation (correlation coefficient=.709; P<.0001). On the other hand, cumulative addition of ACh induced contractions and only a slight increase in [Ca²⁺]_i after stimulation with 0.3 µmol/L PE in aortic strips without endothelium (Fig 1B, top). Cumulative addition of NTG induced decreases in [Ca²⁺]_i and relaxations in both aortic strips with and without endothelium (Fig 1A and 1B, bottom). Furthermore, these fluorescence changes due to ACh and NTG began slightly (<1 second) earlier than the relaxation in the strips precontracted with PE.

View larger version (18K): [in this window] [in a new window]   Figure 1. Typical recordings of [Ca2+]i (indicated by F340/F380; upper trace) and muscle tension (lower trace) in response to ACh and NTG in aortic strips with (A) or without (B) endothelium. Aortic strips were contracted with PE (0.3 µmol/L) and relaxed by cumulative concentrations of ACh or NTG. Concentrations of all drugs are expressed as negative logarithms.

View larger version (11K): [in this window] [in a new window]   Figure 2. Relationship between [Ca2+]i (abscissa) and relaxation (ordinate) induced by ACh in PE-stimulated aortic strips with endothelium. Aortic strips with endothelium were contracted with PE (0.3 µmol/L) and relaxed by cumulative concentrations of ACh. Relaxation values are expressed as percent decreases of the PE-induced response. [Ca2+]i values were expressed as percent values of ACh (3 µmol/L, maximally effective dose)-induced response. The concentration of ACh is indicated by numbers (1, 0.1 µmol/L; 2, 0.3 µmol/L; 3, 1 µmol/L; 4, 3 µmol/L). Each point represents the mean of 9 to 12 experiments. SEM is shown by vertical and horizontal bars. The regression line was drawn by the least-squares method (correlation coefficient=.709, P<.0001).

Effect of Phospholipids on ACh-Induced Relaxation and Increase in [Ca²⁺]_i in Endothelial Cells
Fig 3 demonstrates typical recordings of strips with endothelium preincubated with LPC or PC. We confirmed in the previous observation that LPC or PC by itself altered neither the resting tension nor PE-evoked contraction in aortic strips. Reduced relaxation to ACh was shown in the strips after preincubation with LPC. Fig 4 shows the concentration-response relations of muscle tension for ACh. The preincubation of strips with LPC (2, 10, and 20 µmol/L) shifted the curve to the right and significantly inhibited endothelium-dependent relaxation to ACh in a dose-dependent manner. In contrast, PC (20 µmol/L) had no effect on relaxation to ACh (Figs 3 and 4). As shown in Fig 3, PC alone induced no increase in [Ca²⁺]_i in endothelial cells and LPC (20 µmol/L) by itself slightly increased [Ca²⁺]_i to 143±8 nmol/L in endothelial cells. After preincubation with LPC (20 µmol/L), ACh-evoked rise in [Ca²⁺]_i was completely abolished. LPC (2, 10, and 20 µmol/L) dose dependently inhibited increase in endothelial [Ca²⁺]_i in response to a maximally effective dose of ACh (3 µmol/L) (Fig 5). On the other hand, PC (20 µmol/L) had no effect on ACh-induced increase in [Ca²⁺]_i in endothelial cells (Figs 3 and 5).

View larger version (16K): [in this window] [in a new window]   Figure 3. Typical recordings of [Ca2+]i (indicated by F340/F380; upper trace) and muscle tension (lower trace) in response to ACh in aortic strips with endothelium preincubated with LPC (20 µmol/L; A) or PC (20 µmol/L; B). Aortic strips were contracted with PE (0.3 µmol/L) and relaxed by cumulative concentrations of ACh. After incubation with LPC or PC for 20 minutes, the contraction and relaxation cycle was repeated. Concentrations of ACh are expressed as negative logarithms. w indicates washout.

View larger version (15K): [in this window] [in a new window]   Figure 4. Concentration-response curves showing the effects of PC or LPC on ACh-evoked relaxations in rabbit aortic strips with endothelium. Aortic strips with endothelium were contracted with PE (0.3 µmol/L) in the presence or absence of phospholipids and relaxed by cumulative concentrations of ACh. Relaxations are expressed as percent decreases of the PE-induced precontraction. Control, no phospholipids (, n=12); +PC (20 µmol/L; , n=6); +LPC (2 µmol/L; , n=5); +LPC (10 µmol/L; , n=5); +LPC (20 µmol/L; , n=6). Each point represents the mean values, and SEM is shown by vertical bars. The concentration of ACh is expressed as a negative logarithm. *P<.05; P<.001 vs control value.

View larger version (32K): [in this window] [in a new window]   Figure 5. Effects of phospholipids on ACh (3 µmol/L)-induced relaxation (A) and increase in [Ca2+]i (B) in endothelial cells. Aortic strips with endothelium were contracted with PE (0.3 µmol/L) and relaxed by cumulative concentrations of ACh. After incubation with a selected concentration of phospholipids for 20 minutes, the contraction and relaxation cycle was repeated. Relaxation values induced by ACh (3 µmol/L) are expressed as percent decreases of the PE-induced precontraction and adopted from Fig 4. [Ca2+]i values induced by ACh (3 µmol/L) are expressed as percent values of those before treatment with phospholipids. Data are expressed as mean±SEM of 5 to 12 experiments. *P<.01; P<.001 vs control value.

Substance P, another endothelium-dependent relaxant, induced Ca²⁺ transients in endothelial cells simultaneously with relaxation in the strips with endothelium. Substance P–induced Ca²⁺ transients in endothelial cells and relaxation were also inhibited by LPC (data not shown).

Effect of LPC on NTG-Induced Relaxation and Decrease in [Ca²⁺]_i in Smooth Muscle Cells
LPC or PC by itself altered neither the resting tension nor [Ca²⁺]_i and had no effect on PE-induced contraction and [Ca²⁺]_i in smooth muscle cells in aortic strips without endothelium (data not shown). Fig 6 shows concentration-response relations for NTG in the strips without endothelium. LPC did not affect either NTG-induced endothelium-independent relaxation or decrease in [Ca²⁺]_i in smooth muscle cells.

View larger version (14K): [in this window] [in a new window]   Figure 6. Concentration-response curves showing the effects of LPC on NTG-induced relaxation (A) and decrease in [Ca2+]i in smooth muscle cells (B). Aortic strips without endothelium were contracted with PE (0.3 µmol/L) and relaxed by cumulative concentrations of NTG in the presence () or absence () of LPC (20 µmol/L). Relaxations and [Ca2+]i values are expressed as percent decreases of the PE (0.3 µmol/L)-induced response. Each point represents the mean of 7 experiments, and SEM is shown by vertical bars. The concentration of NTG is expressed as a negative logarithm.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, we demonstrated that (1) LPC simultaneously inhibited both endothelium-dependent relaxation and increase in endothelial [Ca²⁺]_i induced by ACh and (2) LPC affected neither NTG-induced endothelium-independent relaxation nor decrease in [Ca²⁺]_i in smooth muscle cells. These findings suggest that the inhibitory effect of LPC on endothelium-dependent vascular relaxation by ACh is due to the inhibition of ACh-induced Ca²⁺ mobilization in vascular endothelial cells, which is an essential step in the production of EDRF.

Numerous studies have confirmed that vascular endothelium modulates smooth muscle tone by releasing EDRF(s).^{22 23} Several reports demonstrated that the impaired endothelium-dependent relaxation may play an important role in the altered regulation of vascular tone in atherosclerotic arteries.^{1 2 3 4} We also have reported that endothelium-dependent relaxation induced by ACh was impaired in atherosclerotic arteries isolated from Watanabe heritable hyperlipidemic rabbits and that the impairment was further increased during the progression of atherosclerotic plaque formation.⁵ In atherosclerotic aorta, severalfold increases in LPC content were demonstrated in nutritionally induced atherosclerosis.²⁴ Furthermore, ox-LDL has been shown to be present in atherosclerotic arterial lesions in humans and rabbits.⁸ In ox-LDL, 40% of PC is converted to LPC during full oxidative modification.¹⁰ According to these observations, LPC may be an important factor for impairment of endothelium-dependent relaxation in atherosclerotic arteries.

Recently, simultaneous measurement of [Ca²⁺]_i with muscle tension has been performed in intact smooth muscle.^{25 26 27 28} Sato et al¹⁸ have measured endothelial and/or smooth muscle [Ca²⁺]_i simultaneously with muscle tension in rat aorta. An advantage of this method is that it enables us to examine intracellular Ca²⁺ regulation in intact native endothelial cells. In contrast to the case of cultured endothelial cells, cell-surface receptor and signal-transduction systems as well as intercellular communication between endothelial cells and smooth muscle cells remain intact in isolated native endothelial cells without enzymatic or mechanical treatments and culture passages. As another advantage of the present method, the temporal as well as quantitative relationship between [Ca²⁺]_i and vascular function can be examined. In the present experiments it was found that ACh induced additional increases in [Ca²⁺]_i, which preceded the endothelium-dependent relaxation in the strips precontracted with PE. Moreover, we demonstrated that there was a positive correlation between the increase in [Ca²⁺]_i and vascular relaxation. It is possible that ACh-induced increases in endothelial [Ca²⁺]_i obtained from aortic strips with endothelium (which also contain smooth muscle cells) may be underestimated, because ACh decreases [Ca²⁺]_i in smooth muscle cells by EDRF released from endothelium. It may be concluded that the ACh-induced additional increase in [Ca²⁺]_i is due to the increase in endothelial [Ca²⁺]_i and that synthesis of EDRF is regulated by the amount of [Ca²⁺]_i in the endothelial cells.

In the present study, using the strips without endothelium, we confirmed that LPC affected neither resting tension nor NTG-induced endothelium-independent relaxation. Moreover, resting [Ca²⁺]_i and NTG-induced decrease in [Ca²⁺]_i in smooth muscle cells were not affected by LPC. These findings suggest that LPC has no effect on intracellular Ca²⁺ regulation in smooth muscle cells and susceptibility of smooth muscle in response to nitric oxide as EDRF.

It is known that LPC also has nonspecific detergent-like cytotoxic properties. Because the critical micelle concentration of LPC in a physiological solution is reported to be 40 to 50 µmol/L,²⁹ this inhibitory effect might be due to cell lysis by the detergent actions of LPC micelles on cell membrane. The inhibitory effect of LPC was abolished by washing strips with PSS containing 0.1% bovine serum albumin (data not shown). Therefore, the observed effect of LPC was not due to endothelial cell lysis, and the endothelial cell function may be reversibly altered by LPC.

To the best of our knowledge, the findings of this study are the first direct evidence that the inhibition of receptor-mediated Ca²⁺ mobilization in endothelial cells is involved in the inhibitory effect of LPC on endothe- lium-dependent relaxation. Recently, Ohara et al³⁰ showed that activation of protein kinase C by LPC leads to increased O₂^- formation in intact rabbit aorta. Ca²⁺-independent mechanisms, such as inactivation of EDRF by increased O₂^-, may play a role in the inhibition of endothelium-dependent relaxation after exposure to LPC. In this study, however, LPC inhibited both the change in endothelial [Ca²⁺]_i and the change in endothelium-dependent relaxation in parallel. This finding indicates that alteration of endothelial Ca²⁺ regulation may play a main role in the mechanism of LPC-induced impairment of endothelium-dependent vascular relaxation.

The mechanism by which LPC inhibits receptor-mediated Ca²⁺ mobilization in endothelial cells is unclear, because the precise mechanism of the Ca²⁺ influx is not fully established. LPC is an amphiphilic compound and alters the cell function, ie, the kinetics of transmembrane ion transport,³¹ the activities of membrane-bound enzymes,^{32 33 34 35} ligand-receptor coupling,³⁶ and gene expressions.^{37 38 39} According to the previous studies, an increase in LPC may alter physical properties of the plasma membrane, such as membrane fluidity and permeability.⁴⁰ This alteration of membrane fluidity may also displace boundary lipids around integral proteins such as membrane-bound receptors and many G protein effector systems, which might in turn interfere with protein structure and enzymatic activities.⁴¹ The inhibitory effect of LPC may possibly result from the direct interaction with the plasma membrane of endothelial cells. We have reported that LPC and ox-LDL inhibit bradykinin-induced inositol 1,4,5-triphosphate formation and intracellular Ca²⁺ transients in cultured bovine aortic endothelial cells.^{17 42} Moreover, Flavahan⁴³ has recently suggested that LPC may mediate in part the dysfunction in the endothelial G_i protein–dependent pathway. It is possible that an increased incorporation of LPC into plasma membrane of endothelial cells may induce the disruption of the receptor signal-transduction system, leading to the impaired production of EDRF. We confirmed that LPC also inhibited substance P–induced Ca²⁺ transients in endothelial cells and relaxation. This observation suggests that the action of LPC is not as a muscarinic receptor antagonist at endothelial cells.

Recent studies have shown that a hyperpolarization caused by the opening of Ca²⁺-activated K⁺ channels is part of the endothelial response to various receptor agonists.^{44 45 46} This hyperpolarization augments the driving force for the potential-dependent Ca²⁺ influx,^{47 48} not via voltage-gated Ca²⁺ channels,⁴⁵ thereby contributing to the sustained increase in [Ca²⁺]_i. Therefore, depolarization attenuates Ca²⁺ entry into activated endothelial cells.^{45 48} It is also possible that LPC may depolarize endothelial cells, leading to the inhibited Ca²⁺ influx evoked by receptor agonists. The mechanisms of the inhibitory effect of LPC on receptor-mediated Ca²⁺ mobilization need to be clarified in future studies.

In conclusion, LPC, which accumulates in ox-LDL and atherosclerotic arteries, inhibits receptor-mediated Ca²⁺ mobilization in intact aortic endothelial cells of isolated arterial strips. This action of LPC may play an important role in the mechanism of LPC-induced impairment of endothelium-dependent vascular relaxation.

	Selected Abbreviations and Acronyms

 

ACh = acetylcholine [Ca2+]i = cytosolic [Ca2+] EDRF = endothelium-derived relaxing factor LPC = lysophosphatidylcholine NTG = nitroglycerin ox-LDL = oxidized LDL PC = phosphatidylcholine PE = phenylephrine PSS = physiological salt solution

	Acknowledgments

 
This study was supported by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture and for research on cardiovascular disease from the Ministry of Health and Welfare of Japan and from Kanae Foundation of Research for New Medicine and Research for Molecular Cardiology.

Received April 30, 1996; accepted October 25, 1996.

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