- Email: [email protected]
Burst production of superoxide anion in human endothelial cells by lysophosphatidylcholine
Atherosclerosis 143 (1999) 201 – 204
Rapid communication
Burst production of superoxide anion in human endothelial cells by lysophosphatidylcholine Kiyotaka Kugiyama *, Seigo Sugiyama, Nobuhiko Ogata, Hideki Oka, Hideki Doi, Yasutaka Ota, Hirofumi Yasue Di6ision of Cardiology, Kumamoto Uni6ersity School of Medicine, Honjo 1 -1 -1, Kumamoto, 860 -8556 Japan Received 19 May 1998; received in revised form 24 August 1998; accepted 20 October 1998
Abstract This study examined whether lysophosphatidylcholine (lysoPC), an atherogenic lipid, may stimulate production of O2− in cultured human endothelial cells. Production of O2− was detected by bis-N-methylacridinium nitrate (lucigenin)-elicited chemiluminescence. LysoPC was found to induce burst production of O2− , peaked at 24 min after the stimulation, in intact endothelial cells. LysoPC also stimulated NADH-dependent production of O2− in particulate fraction of the cells, and the action of lysoPC was inhibited by diphenyliodonium. The results suggested that lysoPC stimulated production of O2− partly through membrane-associated NADH-dependent O2− production systems. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Free radicals; Endothelium; Lipids
1. Introduction Lysophosphatidylcholine (lysoPC), which accumulates in oxidized low-density lipoproteins (LDL) and in atherosclerotic arterial walls, plays an important role in the alteration of endothelial functions in atherosclerotic arteries [1,2]. We and others have previously shown that lysoPC induced impairment of endothelium-dependent arterial relaxation, upregulation of adhesion molecules, and activation of transcriptional factors in endothelial cells [1– 4]. However, the mechanism for these diverse effects of lysoPC on endothelial functions remains unknown. Reactive oxygen species (ROS) generated in atherosclerotic arterial walls have been shown to cause impairment of endothelium-dependent vasodilation, up-
* Corresponding author. Tel.: +81-96-3735175; fax: + 81-963623256; e-mail: [email protected].
regulation of adhesion molecules, and activation of redox-sensitive transcription factors including nuclear factor-kB (NF-kB) [4–7]. Thus, most of vascular effects of lysoPC are also inducible by ROS. This study examined whether lysoPC could stimulate production of O2− in cultured human endothelial cells.
2. Methods
2.1. Cell culture Primary cultures of endothelial cells from human umbilical vein were obtained in the same manner as we previously described [4]. The confluent endothelial cells were detached by trypsinization and washed three times by Hanks’ balanced salt solution (HBSS). Cultured endothelial cells suspended in HBSS were used for measurement of chemiluminescence responses in intact cells.
0021-9150/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 9 8 ) 0 0 2 8 8 - 3
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K. Kugiyama et al. / Atherosclerosis 143 (1999) 201–204
2.2. Fractionation of cultured endothelial cells The confluent endothelial cells were washed three times with phosphate-buffered saline (PBS) and scraped in cold HBSS with protease inhibitors. The cells were then disrupted by sonication (sonifier 250, Bronson) at 4°C. The suspension was then centrifuged at 800×g for 10 min at 4°C to remove unbroken cells and nuclear materials. Then, the postnuclear supernatant was centrifuged at 100 000× g for 60 min for separation into cytosolic and particulate fractions. The pellet (particulate fraction) was resuspended in cold HBSS with the protease inhibitors and again sonicated at 4°C.
2.3. Measurements of superoxide anion production Production of O2− was detected by chemiluminescence of bis-N-methylacridinium nitrate (lucigenin) by use of luminescence reader (BLR-201, Aloka, Tokyo, Japan) [5,8,9]. The system using the lucigenin-chemiluminescence detected O2− generated by xanthine (0.5 mM)/xanthine oxidase (0.03 U/ml) and by neutrophils activated by N-formyl-methionyl-leucyl-phenylalanine (0.1 mM), and the chemiluminescence was completely suppressed by superoxide dismutase (SOD; 300 U/ml) and 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron;10 mM) but neither catalase (1000 U/ml) nor mannitol (5 mM). Further, this system had no significant response of the chemiluminescence to H2O2 (0.5 mM) and human myeloperoxidase (Sigma, M6908; 10 mg/ml) in cell free assay buffer. Thus, the lucigenin chemiluminescence used in this study is an adequate method for detection of O2− production, as established by a number of previous studies [5,8,9]. The suspended endothelial cells were mixed with HBSS containing Cu2 + (1.3 mM), Mg2 + (0.4 mM), and lucigenin (250 mM) at 37°C in a total volume of 1 ml (1× 106 cells) in the presence or absence of various inhibitors. Counts were performed at baseline and every 30 s until 15 min after addition of lysoPC into the assay mixture. The b-Nicotinamide adenine dinucleotide, reduced form (NADH)- and b-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-dependent generation of O2− in particulate and cytosolic fractions was also measured by the chemiluminescence in HBSS containing Cu2 + (1.3 mM), Mg2 + (0.4 mM), lucigenin (250 mM), cell protein (100 mg), and NADH or NADPH (100 mM at final concentration) at 37°C in a total volume of 1 ml in the presence or absence of various inhibitors, and the counts were measured every 30 s until 20 min after addition of cell protein. The chemiluminescence response was estimated by subtraction of the chemiluminescence in the absence of cells and cell fractions and was standardized using a standard curve generated from known quantities of xanthine and xanthine oxidase. A chemiluminescence signal
of 500 cpm was approximately equivalent to a rate of 1 nmol/min of O2− generation in our system.
2.4. Materials Rotenone and diphenyliodonium (DPI) were from Aldrich (Milwaukee, MI) and N,N-diethylaminoethyl2,2-diphenylvalerate hydrochloride (SKF-525A) was from RBI (Boston, MA). Tiron (D7389), SOD (S5639), 2-[12-Hydroxydodeca-5,10-diynyl] -3,5,6-trimethyl-p-benzoquinone (AA861, A3711), oxypurinol, N w-nitro-L-arginine methyl ester (L-NAME, N1522), indomethacin (I7378), lucigenin (M8010), NADH (N8129), NADPH (N1630), catalase (C40), xanthine (X0626), xanthine oxidase (X4875), and other chemicals were obtained from Sigma (St. Louis, Mo).
2.5. Statistical analysis All values were expressed as mean 9 S.E.M. Statistical evaluation of data was performed by Student’s t-test for unpaired observations. When more than two groups were compared, one-way analysis of variance (ANOVA) following the Fisher’s protected least significant difference test was used. A value of PB 0.05 was considered significant.
3. Results
3.1. Production of O 2− in intact endothelial cells The addition of a-palmitoyl-L-lysoPC (C16:0) into the incubation mixture stimulated O2− production in intact endothelial cells with a lag phase of approximately 1 min, and the O2− production was peaked at 2 4 min after the addition of lysoPC and was subse-
Fig. 1. Time course of lucigenin-elicited chemiluminescence in intact human endothelial cells after addition of palmitoyl lysoPC (7.5 mM) or same volume of PBS as a vehicle into the assay mixture. Data are mean of two independent experiments.
K. Kugiyama et al. / Atherosclerosis 143 (1999) 201–204 Table 1 Effects of various inhibitors on lysoPC-induced production of O− 2 in intact human endothelial cellsa Chemiluminescence (cpm/1×106 cells) Basal
60 92
LysoPC (palmitoyl, 7.5 mM) +DPI (10 mM) +rotenone (100 mM) +SKF-525A (200 mM) +Tiron (10 mM) +SOD (1000 U/ml) +oxypurinol (100 mM) +indomethacin (10 mM) +L-NAME (50 mM) +AA861 (1 mM)
11209 70 710 9 20* 820 9 60* 720940* 1909 10* 1050 960 1150930 12609 60 10809 40 1180950
a Each value is mean9 S.E.M of 68 experiments. Lucigenin-elicited chemiluminescence was continuously measured after addition of palmitoyl lysoPC (7.5 mM) into the assay mixture containing intact human endothelial cells (1×106 cells/ml) and one of the inhibitors. Chemiluminescence response was estimated at its peak. These inhibitors had no direct ‘quenching’ effect on the lucigenin chemiluminescence. * PB0.01 vs. lysoPC alone.
quently decreased to the baseline level within 40 min after the stimulation (Fig. 1). The effect of lysoPC was dependent on its concentrations; low concentrations of lysoPC (5–15 mM) stimulated production of O2− , while the higher concentrations (3050 mM, maximal concentrations tested) had minimal effect. The incubation of the endothelial cells with lysoPC at the higher concentrations of up to 50 mM for 30 min did not significantly release lactate dehydrogenase from endothelial cells into the culture medium (data not shown) as observed in our previous papers [3,4], suggesting that the effect of lysoPC at the higher concentrations was unlikely to be due to its non specific cytotoxicity. a-Stearoyl-L-lysoPC (C18:0, 7.5 mM) also stimulated O2− production (9809 30 cpm/1 ×106 cells at peak) in a similar manner as a-palmitoyl-L-lysoPC, but dipalmitoyl phosphatidylcholine (530 mM) had no effect. Combined incubation of endothelial cells with Tiron (10 mM), a low molecular-weight and cell-permeable scavenger of O2− , completely abolished lysoPC-induced production of O2− , while SOD (1000 U/ml) had little effect (Table 1). Thus, O2− was mainly produced by intracellular mechanisms. LysoPC-induced production of O2− was inhibited by combined incubation of endothelial cells with DPI, an inhibitor of flavin-contained enzymes, rotenone, an inhibitor of mitochondrial cytochrome oxidases, and SKF-525A, an inhibitor of microsomal oxidases (Table 1). However, L-NAME, oxypurinol, indomethacin, and AA861 did not signifi-
203
cantly affect lysoPC-induced production of O2− (Table 1).
3.2. Production of O 2− in cell fractions The addition of particulate fraction into the assay mixture caused production of O2− in the presence of NADH or NADPH (Fig. 2), while it had no production of O2− in the absence of NADH and NADPH. The production of O2− in particulate fraction was increased gradually and reached plateau within 20 min after the addition of cell protein. Thus, chemiluminescence response in particulate fraction was estimated at 20 min after the addition of cell protein. The NADHand NADPH-dependent productions of O2− in cytosolic fraction were both minimal (both less than 5% of those in particular fraction). Presence of lysoPC in the assay mixture augmented NADH- and NADPH-dependent production of O2− in particulate fraction (Fig. 2). However, the extent of lysoPC-induced augmentation of O2− production in particulate fraction was greater in response to NADH than that to NADPH (Fig. 2). The addition of SOD (300 U/ml) into the assay buffer abolished NADH- and NADPH-dependent increase in the lucigenin chemiluminescence in either presence or absence of lysoPC by more than 90%. DPI (10 mM), rotenone (100 mM), and SKF-525A (100 mM) significantly suppressed lysoPC-induced augmentation of NADH-dependent production of O2− in particulate fraction by 42 93, 52 95 and 39 9 6%, respectively.
Fig. 2. Effects of palmitoyl lysoPC on NADH- and NADPH-dependent production of O2− in particulate fraction of endothelial cells. Particulate fraction (100 mg protein) was added into the assay mixture (1 ml) with NADH (100 mM) or NADPH (100 mM) in the presence or absence of palmitoyl lysoPC (10 mM). Chemiluminescence response in particulate fraction was continuously measured and estimated at 20 min after the addition of cell protein. Each value is mean 9S.E.M. of six experiments.
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4. Discussion
References
This is the first report to show that lysoPC stimulated production of O2− in endothelial cells. The addition of lysoPC in intact cell suspensions induced the burst generation of O2− through intracellular mechanisms. Further, the present study showed that lysoPC stimulated NADH-dependent production of O2− in particulate fraction. These results and the experiments with use of various inhibitors suggested that membrane-associated NADH-dependent O2− production systems, partly in mitochondrial and microsomal fractions, may be probably responsible for the lysoPC-induced production of O2− in intact endothelial cells. Production of intracellular O2− in the cells treated by lysoPC was supported by our preliminary data of the increase in formazan-positive cells treated by LysoPC using nitroblue tetrazolium reduction assay [10] (unpublished data, 1998). LysoPC abundantly exists in extracellular space of intima in atherosclerotic arteries [11]. Also, lysoPC is known to be generated in intracellular space by cytosolic phospholipase A2 which plays an important role in the intracellular signal transduction [12]. Thus, the present study suggested that lysoPC derived from either extracellular or intracellular source could produce O2− in endothelial cells of atherosclerotic arteries. There is a possibility that some biological effects of lysoPC on endothelial functions could be mediated by O2− . In conclusion, lysoPC induced burst generation of O2− in endothelial cells through intracellular mechanisms. Membrane-associated NADH-dependent O2− production systems may be partly responsible for the lysoPC-induced production of O2− in endothelial cells.
[1] Kugiyama K, Kerns SA, Moriset JD, Roberts R, Henry PD. Impairment of endothelium-dependent arterial relaxation by lysolecithin in modified low-density lipoproteins. Nature 1990;344:160 – 2. [2] Witztum JL, Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest 1991;88:1785 – 92. [3] Sugiyama S, Kugiyama K, Ohgushi M, Fujimoto K, Yasue H. Lysophosphatidylcholine in oxidized low-density lipoprotein increases endothelial susceptibility to polymorphonuclear leukocyte-induced endothelial dysfunction in porcine coronary arteries: role of protein kinase C. Circ Res 1994;74:565–75. [4] Sugiyama S, Kugiyama K, Oagata N, et al. Biphasic regulation of transcription factor nuclear factor-kB activity in human endothelial cells by lysophosphatidylcholine through protein kinase C-mediated pathway. Arterioscler Thromb Vasc Biol 1998;18:568 – 76. [5] Ohara Y, Peterson TE, Harrison DG. Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest 1993;91:2546 – 51. [6] Marui N, Offermann MK, Swerlick R, et al. Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells. J Clin Invest 1993;92:1866–74. [7] Collins T. Endothelial nuclear factor-kappa B and the initiation of the atherosclerotic lesion. Lab Invest 1993;68:499 – 508. [8] Gyllenhammar H. Lucigenin chemiluminescence in the assessment of neutrophil superoxide production. J Immunol Methods 1987;97:209 – 13. [9] Mohazzab-H KM, Kaminski PM, Wolin MS. NADH oxidoreductase is a major source of superoxide anion in bovine coronary artery endothelium. Am J Physiol 1994;266:2568 – 72. [10] DiGregorio KA, Cilento EV, Lantz RC. Measurement of superoxide release from single pulmonary alveolar macrophages. Am J Physiol 1987;252:677 – 83. [11] Portman OW, Soltys P, Alexander M, Osuga T. Metabolism of lysolecithin in vivo: effects of hyperlipidemia and atherosclerosis in squirrel monkeys. J Lipid Res 1970;2:596 – 604. [12] Exton JH. Phosphatidylcholine breakdown and signal transduction. Biochem Biophys Acta 1994;1212:26 – 42.
.
Rapid communication
Burst production of superoxide anion in human endothelial cells by lysophosphatidylcholine Kiyotaka Kugiyama *, Seigo Sugiyama, Nobuhiko Ogata, Hideki Oka, Hideki Doi, Yasutaka Ota, Hirofumi Yasue Di6ision of Cardiology, Kumamoto Uni6ersity School of Medicine, Honjo 1 -1 -1, Kumamoto, 860 -8556 Japan Received 19 May 1998; received in revised form 24 August 1998; accepted 20 October 1998
Abstract This study examined whether lysophosphatidylcholine (lysoPC), an atherogenic lipid, may stimulate production of O2− in cultured human endothelial cells. Production of O2− was detected by bis-N-methylacridinium nitrate (lucigenin)-elicited chemiluminescence. LysoPC was found to induce burst production of O2− , peaked at 24 min after the stimulation, in intact endothelial cells. LysoPC also stimulated NADH-dependent production of O2− in particulate fraction of the cells, and the action of lysoPC was inhibited by diphenyliodonium. The results suggested that lysoPC stimulated production of O2− partly through membrane-associated NADH-dependent O2− production systems. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Free radicals; Endothelium; Lipids
1. Introduction Lysophosphatidylcholine (lysoPC), which accumulates in oxidized low-density lipoproteins (LDL) and in atherosclerotic arterial walls, plays an important role in the alteration of endothelial functions in atherosclerotic arteries [1,2]. We and others have previously shown that lysoPC induced impairment of endothelium-dependent arterial relaxation, upregulation of adhesion molecules, and activation of transcriptional factors in endothelial cells [1– 4]. However, the mechanism for these diverse effects of lysoPC on endothelial functions remains unknown. Reactive oxygen species (ROS) generated in atherosclerotic arterial walls have been shown to cause impairment of endothelium-dependent vasodilation, up-
* Corresponding author. Tel.: +81-96-3735175; fax: + 81-963623256; e-mail: [email protected].
regulation of adhesion molecules, and activation of redox-sensitive transcription factors including nuclear factor-kB (NF-kB) [4–7]. Thus, most of vascular effects of lysoPC are also inducible by ROS. This study examined whether lysoPC could stimulate production of O2− in cultured human endothelial cells.
2. Methods
2.1. Cell culture Primary cultures of endothelial cells from human umbilical vein were obtained in the same manner as we previously described [4]. The confluent endothelial cells were detached by trypsinization and washed three times by Hanks’ balanced salt solution (HBSS). Cultured endothelial cells suspended in HBSS were used for measurement of chemiluminescence responses in intact cells.
0021-9150/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 9 8 ) 0 0 2 8 8 - 3
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K. Kugiyama et al. / Atherosclerosis 143 (1999) 201–204
2.2. Fractionation of cultured endothelial cells The confluent endothelial cells were washed three times with phosphate-buffered saline (PBS) and scraped in cold HBSS with protease inhibitors. The cells were then disrupted by sonication (sonifier 250, Bronson) at 4°C. The suspension was then centrifuged at 800×g for 10 min at 4°C to remove unbroken cells and nuclear materials. Then, the postnuclear supernatant was centrifuged at 100 000× g for 60 min for separation into cytosolic and particulate fractions. The pellet (particulate fraction) was resuspended in cold HBSS with the protease inhibitors and again sonicated at 4°C.
2.3. Measurements of superoxide anion production Production of O2− was detected by chemiluminescence of bis-N-methylacridinium nitrate (lucigenin) by use of luminescence reader (BLR-201, Aloka, Tokyo, Japan) [5,8,9]. The system using the lucigenin-chemiluminescence detected O2− generated by xanthine (0.5 mM)/xanthine oxidase (0.03 U/ml) and by neutrophils activated by N-formyl-methionyl-leucyl-phenylalanine (0.1 mM), and the chemiluminescence was completely suppressed by superoxide dismutase (SOD; 300 U/ml) and 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron;10 mM) but neither catalase (1000 U/ml) nor mannitol (5 mM). Further, this system had no significant response of the chemiluminescence to H2O2 (0.5 mM) and human myeloperoxidase (Sigma, M6908; 10 mg/ml) in cell free assay buffer. Thus, the lucigenin chemiluminescence used in this study is an adequate method for detection of O2− production, as established by a number of previous studies [5,8,9]. The suspended endothelial cells were mixed with HBSS containing Cu2 + (1.3 mM), Mg2 + (0.4 mM), and lucigenin (250 mM) at 37°C in a total volume of 1 ml (1× 106 cells) in the presence or absence of various inhibitors. Counts were performed at baseline and every 30 s until 15 min after addition of lysoPC into the assay mixture. The b-Nicotinamide adenine dinucleotide, reduced form (NADH)- and b-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-dependent generation of O2− in particulate and cytosolic fractions was also measured by the chemiluminescence in HBSS containing Cu2 + (1.3 mM), Mg2 + (0.4 mM), lucigenin (250 mM), cell protein (100 mg), and NADH or NADPH (100 mM at final concentration) at 37°C in a total volume of 1 ml in the presence or absence of various inhibitors, and the counts were measured every 30 s until 20 min after addition of cell protein. The chemiluminescence response was estimated by subtraction of the chemiluminescence in the absence of cells and cell fractions and was standardized using a standard curve generated from known quantities of xanthine and xanthine oxidase. A chemiluminescence signal
of 500 cpm was approximately equivalent to a rate of 1 nmol/min of O2− generation in our system.
2.4. Materials Rotenone and diphenyliodonium (DPI) were from Aldrich (Milwaukee, MI) and N,N-diethylaminoethyl2,2-diphenylvalerate hydrochloride (SKF-525A) was from RBI (Boston, MA). Tiron (D7389), SOD (S5639), 2-[12-Hydroxydodeca-5,10-diynyl] -3,5,6-trimethyl-p-benzoquinone (AA861, A3711), oxypurinol, N w-nitro-L-arginine methyl ester (L-NAME, N1522), indomethacin (I7378), lucigenin (M8010), NADH (N8129), NADPH (N1630), catalase (C40), xanthine (X0626), xanthine oxidase (X4875), and other chemicals were obtained from Sigma (St. Louis, Mo).
2.5. Statistical analysis All values were expressed as mean 9 S.E.M. Statistical evaluation of data was performed by Student’s t-test for unpaired observations. When more than two groups were compared, one-way analysis of variance (ANOVA) following the Fisher’s protected least significant difference test was used. A value of PB 0.05 was considered significant.
3. Results
3.1. Production of O 2− in intact endothelial cells The addition of a-palmitoyl-L-lysoPC (C16:0) into the incubation mixture stimulated O2− production in intact endothelial cells with a lag phase of approximately 1 min, and the O2− production was peaked at 2 4 min after the addition of lysoPC and was subse-
Fig. 1. Time course of lucigenin-elicited chemiluminescence in intact human endothelial cells after addition of palmitoyl lysoPC (7.5 mM) or same volume of PBS as a vehicle into the assay mixture. Data are mean of two independent experiments.
K. Kugiyama et al. / Atherosclerosis 143 (1999) 201–204 Table 1 Effects of various inhibitors on lysoPC-induced production of O− 2 in intact human endothelial cellsa Chemiluminescence (cpm/1×106 cells) Basal
60 92
LysoPC (palmitoyl, 7.5 mM) +DPI (10 mM) +rotenone (100 mM) +SKF-525A (200 mM) +Tiron (10 mM) +SOD (1000 U/ml) +oxypurinol (100 mM) +indomethacin (10 mM) +L-NAME (50 mM) +AA861 (1 mM)
11209 70 710 9 20* 820 9 60* 720940* 1909 10* 1050 960 1150930 12609 60 10809 40 1180950
a Each value is mean9 S.E.M of 68 experiments. Lucigenin-elicited chemiluminescence was continuously measured after addition of palmitoyl lysoPC (7.5 mM) into the assay mixture containing intact human endothelial cells (1×106 cells/ml) and one of the inhibitors. Chemiluminescence response was estimated at its peak. These inhibitors had no direct ‘quenching’ effect on the lucigenin chemiluminescence. * PB0.01 vs. lysoPC alone.
quently decreased to the baseline level within 40 min after the stimulation (Fig. 1). The effect of lysoPC was dependent on its concentrations; low concentrations of lysoPC (5–15 mM) stimulated production of O2− , while the higher concentrations (3050 mM, maximal concentrations tested) had minimal effect. The incubation of the endothelial cells with lysoPC at the higher concentrations of up to 50 mM for 30 min did not significantly release lactate dehydrogenase from endothelial cells into the culture medium (data not shown) as observed in our previous papers [3,4], suggesting that the effect of lysoPC at the higher concentrations was unlikely to be due to its non specific cytotoxicity. a-Stearoyl-L-lysoPC (C18:0, 7.5 mM) also stimulated O2− production (9809 30 cpm/1 ×106 cells at peak) in a similar manner as a-palmitoyl-L-lysoPC, but dipalmitoyl phosphatidylcholine (530 mM) had no effect. Combined incubation of endothelial cells with Tiron (10 mM), a low molecular-weight and cell-permeable scavenger of O2− , completely abolished lysoPC-induced production of O2− , while SOD (1000 U/ml) had little effect (Table 1). Thus, O2− was mainly produced by intracellular mechanisms. LysoPC-induced production of O2− was inhibited by combined incubation of endothelial cells with DPI, an inhibitor of flavin-contained enzymes, rotenone, an inhibitor of mitochondrial cytochrome oxidases, and SKF-525A, an inhibitor of microsomal oxidases (Table 1). However, L-NAME, oxypurinol, indomethacin, and AA861 did not signifi-
203
cantly affect lysoPC-induced production of O2− (Table 1).
3.2. Production of O 2− in cell fractions The addition of particulate fraction into the assay mixture caused production of O2− in the presence of NADH or NADPH (Fig. 2), while it had no production of O2− in the absence of NADH and NADPH. The production of O2− in particulate fraction was increased gradually and reached plateau within 20 min after the addition of cell protein. Thus, chemiluminescence response in particulate fraction was estimated at 20 min after the addition of cell protein. The NADHand NADPH-dependent productions of O2− in cytosolic fraction were both minimal (both less than 5% of those in particular fraction). Presence of lysoPC in the assay mixture augmented NADH- and NADPH-dependent production of O2− in particulate fraction (Fig. 2). However, the extent of lysoPC-induced augmentation of O2− production in particulate fraction was greater in response to NADH than that to NADPH (Fig. 2). The addition of SOD (300 U/ml) into the assay buffer abolished NADH- and NADPH-dependent increase in the lucigenin chemiluminescence in either presence or absence of lysoPC by more than 90%. DPI (10 mM), rotenone (100 mM), and SKF-525A (100 mM) significantly suppressed lysoPC-induced augmentation of NADH-dependent production of O2− in particulate fraction by 42 93, 52 95 and 39 9 6%, respectively.
Fig. 2. Effects of palmitoyl lysoPC on NADH- and NADPH-dependent production of O2− in particulate fraction of endothelial cells. Particulate fraction (100 mg protein) was added into the assay mixture (1 ml) with NADH (100 mM) or NADPH (100 mM) in the presence or absence of palmitoyl lysoPC (10 mM). Chemiluminescence response in particulate fraction was continuously measured and estimated at 20 min after the addition of cell protein. Each value is mean 9S.E.M. of six experiments.
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K. Kugiyama et al. / Atherosclerosis 143 (1999) 201–204
4. Discussion
References
This is the first report to show that lysoPC stimulated production of O2− in endothelial cells. The addition of lysoPC in intact cell suspensions induced the burst generation of O2− through intracellular mechanisms. Further, the present study showed that lysoPC stimulated NADH-dependent production of O2− in particulate fraction. These results and the experiments with use of various inhibitors suggested that membrane-associated NADH-dependent O2− production systems, partly in mitochondrial and microsomal fractions, may be probably responsible for the lysoPC-induced production of O2− in intact endothelial cells. Production of intracellular O2− in the cells treated by lysoPC was supported by our preliminary data of the increase in formazan-positive cells treated by LysoPC using nitroblue tetrazolium reduction assay [10] (unpublished data, 1998). LysoPC abundantly exists in extracellular space of intima in atherosclerotic arteries [11]. Also, lysoPC is known to be generated in intracellular space by cytosolic phospholipase A2 which plays an important role in the intracellular signal transduction [12]. Thus, the present study suggested that lysoPC derived from either extracellular or intracellular source could produce O2− in endothelial cells of atherosclerotic arteries. There is a possibility that some biological effects of lysoPC on endothelial functions could be mediated by O2− . In conclusion, lysoPC induced burst generation of O2− in endothelial cells through intracellular mechanisms. Membrane-associated NADH-dependent O2− production systems may be partly responsible for the lysoPC-induced production of O2− in endothelial cells.
[1] Kugiyama K, Kerns SA, Moriset JD, Roberts R, Henry PD. Impairment of endothelium-dependent arterial relaxation by lysolecithin in modified low-density lipoproteins. Nature 1990;344:160 – 2. [2] Witztum JL, Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest 1991;88:1785 – 92. [3] Sugiyama S, Kugiyama K, Ohgushi M, Fujimoto K, Yasue H. Lysophosphatidylcholine in oxidized low-density lipoprotein increases endothelial susceptibility to polymorphonuclear leukocyte-induced endothelial dysfunction in porcine coronary arteries: role of protein kinase C. Circ Res 1994;74:565–75. [4] Sugiyama S, Kugiyama K, Oagata N, et al. Biphasic regulation of transcription factor nuclear factor-kB activity in human endothelial cells by lysophosphatidylcholine through protein kinase C-mediated pathway. Arterioscler Thromb Vasc Biol 1998;18:568 – 76. [5] Ohara Y, Peterson TE, Harrison DG. Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest 1993;91:2546 – 51. [6] Marui N, Offermann MK, Swerlick R, et al. Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells. J Clin Invest 1993;92:1866–74. [7] Collins T. Endothelial nuclear factor-kappa B and the initiation of the atherosclerotic lesion. Lab Invest 1993;68:499 – 508. [8] Gyllenhammar H. Lucigenin chemiluminescence in the assessment of neutrophil superoxide production. J Immunol Methods 1987;97:209 – 13. [9] Mohazzab-H KM, Kaminski PM, Wolin MS. NADH oxidoreductase is a major source of superoxide anion in bovine coronary artery endothelium. Am J Physiol 1994;266:2568 – 72. [10] DiGregorio KA, Cilento EV, Lantz RC. Measurement of superoxide release from single pulmonary alveolar macrophages. Am J Physiol 1987;252:677 – 83. [11] Portman OW, Soltys P, Alexander M, Osuga T. Metabolism of lysolecithin in vivo: effects of hyperlipidemia and atherosclerosis in squirrel monkeys. J Lipid Res 1970;2:596 – 604. [12] Exton JH. Phosphatidylcholine breakdown and signal transduction. Biochem Biophys Acta 1994;1212:26 – 42.
.