BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL

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1 Vol. 44, No. 6, May 1998 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages EFFECTS OF SHORT- AND LONG-TERM EXPOSURE TO L1NOLEIC ACID HYDROPEROXIDE ON CYTOSOLIC CALCIUM ION LEVEL OF HUMAN AORTIC 1NTIMAL SMOOTH MUSCLE CELLS IN VITRO Nobuyuki Arima*, Yasuyuki Sasaguri* and Kunio YagitJ *Department of Pathology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu , Japan; and?institute of Applied Biochemistry, Yagi Memorial Park, Mitake, Gifu , Japan Received December 6, 1997 Summary. When cultured intimal smooth muscle cells from human aorta were exposed for a short period of time (25 sec) to linoleic acid hydroperoxide (100 nmol/ml), influx of calcium ions into the cytosol of these cells was provoked, and a temporal increase in cytosolic calcium ion level was observed. Two calcium channel blockers inhibited this influx. Long-term exposure (4 min) of these cells to the hydroperoxide also provoked the influx of calcium ions, which resulted in a longer time of the calcium ion increase. In the latter case, however, the calcium channel blocker inhibited the initial influx, but then the influx started and continued even in the presence of the blocker. Such difference in exposure time-dependent effects should be taken into account in considering pathological roles of lipid hydroperoxides. Key Words: Linoleic acid hydroperoxide; calcium ion level; aortic intimal smooth muscle cell. INTRODUCTION Pathogenicity of lipid hydroperoxides and related radical species has been well recognized. For example, in atherogenesis, lipid hydroperoxides initiate this disorder by causing injury to endothelial ceils [1, 2], and accelerate its propagation by inhibiting prostacyclin biosynthesis [3-5] and by contributing to the formation of lipid-laden cells, viz., foam cells [6, 7]. In this regard, Hirosumi et al. [8] reported that linoleic acid hydroperoxide (LOOH) elevates the cytosolic calcium ion level in cultured arterial endothelial cells, and suggested the involvement of such elevation in atherogenesis. Abbreviations: LOOH, linoleic acid hydroperoxide; ISMC, intimal smooth muscle cells; DMEM, Dulbecco's modified Eagle's medium. 1 To whom correspondence should be addressed. Fax: E- mail: yagiab@po.infosphere.or.jp /98/ /0 Copyright by Academic Press Australia All rights of reproduction in any form reserved.

BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Recently, we found using cultured arterial endothelial and intimal smooth muscle cells (ISMC) that LOOH stimulated the production of

2 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Recently, we found using cultured arterial endothelial and intimal smooth muscle cells (ISMC) that LOOH stimulated the production of metalloproteinases, which are obviously involved in arterial tissue remodeling, and we thought that the increase in cytosoiic calcium ion level in these cells would be, at least in part, the cause of such stimulation [9, 10]. On the basis of these considerations, we decided to examine in more detail the feature of the increase in calcium ion level in these cells, and found that short term- and long-term exposure to LOOH elevated cytosolic calcium ion level through different mechanisms. This paper briefly reports these results. MATERIALS AND METHODS ISMC. Aortic ISMC were isolated from human aortic intima and cultured as reported previously [9]. Briefly, after the thoracic organs including the aorta had been removed from a cadaver, the adventitia of the aorta was easily peeled off from one end. After removal of endothelial cells from the subendothelial bed by incubation in serum-free Dulbecco's modified Eagle's medium (DMEM, Nissui Pharmaceutical Co., Tokyo) containing 1000 U/ml dispase (Sanko Pharmaceutical Co., Tokyo) for 90 min at 37~ the inner surface was then washed with phosphate-buffered saline to isolate ISMC from the intima. The ISMC were resuspended in DMEM supplemented with 10% t~tal bovine serum together with an antibiotic cocktail consisting of 100 ~tg/ml streptomycin, 100 U/ml penicillin, and 5 ~tg/ml fungizone, and cultured at 37~ under an atmosphere of humidified 5% CO2-95% air mixture. LOOH. LOOH was prepared enzymatically from linoleic acid, obtained from Sigma Chemical Co., St. Louis, MO, with soy bean lipoxygenase purchased from the same company, and purified by thin-layer chromatography with a solvent system of hexane-diethyl ether (8:2, v/v), as described previously [11]. The amount of LOOH was measured by the hemoglobin-methylene blue method [12]. Prior to use, the preparation was dissolved in ethanol to be 40 ~tmol/ml of LOOH. Measurement of calcium ion level in individual cells. Calcium ion level in individual cells was measured as reported previously [10]. Briefly, the cells were incubated for 30 min with fura-2/am (1 ~tm) diluted with the culture medium and then washed with the medium for 10 min. Further, the cells were washed twice with HEPES-buffered saline solution composed of 20 mm HEPES (ph 7.4), 115 mm NaC1, 5.4 mm KC1, 2.2 mm CaC12, 0.8 mm MgCI2, and 13.8 mm glucose. Then, the dish was placed on the thermostated stage of an Olympus IMT-2 inverted microscope. Agents dissolved in the abovementioned HEPES-buffered saline solution were applied to the cells on cover slips by the bath application method. The calcium ion level in individual cells 1188

3 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL was measured fluorometrically. Fluorescence excitation was provided from a Hamamatsu 75W Xe lamp, and excitation wavelengths of 340 and 380 nm were selected by a computer-controlled movement of filters in the light path. Paired recordings were made every 5 sec, and the fluorescence images were obtained with a Hamamatsu SIT camera C , and stored in a digital image processor, Argus-100. The calcium ion level was calculated on a pixel basis from the ratio of the fluorescence intensities obtained with excitation at 340 and 380 nm. Calcium channel blockers. As calcium channel blockers, nicardipine and nifedipine were used. They were purchased from Wako Pure Chem. Ind., Ltd., Osaka. RESULTS AND DISCUSSION Effect of short-term exposure to LOOH on eytosolic calcium ion level By use of digital fluorescence microscopy, the basal calcium ion levels in ISMC were calculated to be around 100 nm, as shown in Fig. 1. When 100 nmol/ml of LOOH was applied to ISMC in vitro for 25 sec and washed out, the cytosolic calcium ion level in individual cells increased immediately and then rapidly decreased, as shown in Fig. 1-A. When the concentration of LOOH was lowered to 60 nmol/ml, the increase in calcium ion level was smaller. The increase after the application of the hydroperoxide was different with different cells. The elevation of cytosolic calcium ion level after the addition of the hydroperoxide mentioned above was not seen when calcium ions were eliminated from the medium, indicating that the increase is due to the influx of calcium ions from the medium into the cells. To assess the involvement of calcium channels in the increase in calcium ion level mentioned above, we used calcium channel blockers, nicardipine and nifedipine. When either blocker was mixed with the medium at its concentration of M, the above-mentioned transient increase in cytosolic calcium ion level was not observed, as shown in Fig. 1-B. This means that the increase in cytosolic calcium ion level in these cells upon exposure to LOOH for a short period of time is ascribable to the influx of calcium ions into the cell through calcium channels. The entering calcium ions would be subjected to cellular calcium ion homeostatic mechanisms, as described by Richter [13 ]. Thus, the temporal increase would be found. 1189

4 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL I000 A 1000 B = E -~ o I Time (sec) Fig. 1. Time course of the change in cytosolic calcium ion level of ISMC exposed to LOOH for a short time. Shadowed area indicates the exposure to LOOH. A. Time course of calcium ion level of each individual cell after the exposure for 25 sec to LOOH. e, exposure to 60 nmol/ml of LOOH; o, that to 100 nmol/ml oflooh. B. Exposure to 100 nmol/ml of LOOH for 25 sec in the presence of 10-6 M nifedipine ( [] ) or 10-6 M nicardipine ( 9 ) ~o 1500 A O E.~ o Time (sec) Fig. 2. Time course of the change in cytosolic calcium ion level of ISMC exposed to LOOH for a long time. Shadowed area indicates the exposure to 100 nmol/ml oflooh. A. Time course of calcium ion level &each individual cell after the exposure for 4 min in the absence of channel blocker. B. Exposure to LOOH for 4 rain in the presence of 10-6 M nifedipine ( o ) or 10-6 M nicardipine ( 9 ). I t90

5 BIOCHEMISTRYGnd MOLECULAR BIOLOGY INTERNATIONAL Effect of long-term exposure to LOOH on cytosolic calcium ion level When ISMC were exposed to 100 nmol/ml of LOOH for 4 min, the cytosolic calcium ion level in all of the 5 cells examined increased. Four cells out of 5 cells began to show an increase in the level immediately after the addition of LOOH, but one cell showed an increase after the end of the exposure period, as shown in Fig. 2-A. Oscillatory elevation was found. The high level was continued for a longer time than that observed with a shorttime exposure. The effects of calcium channel blockers are shown in Fig. 2-B. As can be seen in the figure, the cytosolic calcium ion level was not increased for 3 min after the addition of LOOH, but then it increased to a high level. These results show that for a short-time exposure of the cells to LOOH, the calcium channel blockers were working, but that for a long-time exposure to the hydroperoxide, the incorporation of calcium ions began to occur without the aid of calcium channels. This incorporation would be due to the damage of calcium channels and/or to some route other than calcium channels. It is well known that the cell membrane is damaged to leak lactate dehydrogenase from the cell to the medium upon exposure to LOOH. All these results indicate that the increases in the cytosolic calcium ion level of aortic ISMC lbllowing short- and long-term exposure to LOOH are due to different mechanisms. The former occurs via calcium channels of the cell, whereas the latter might be due to other routes, probably brought into being by damage to the cell membrane. An elevated cytosolic calcium ion level for a longer time would result in cell death. This should be taken into account when we consider the action of lipid hydroperoxides in terms of their pathogenicity. REFERENCES 1. Yagi, K., Ohkawa, H., Ohishi, N., Yamashita, M., and Nakashima, T. (1981) J. Appl. Biochem. 3, Sasaguri, Y., Nakashima, T., Morimatsu, M., and Yagi, K. (1984) J. Appl. Biochem. 6, Moncada, S., Gryglewski, R. J., Bunting, S., and Vane, J. R. (1976) Nature 263, Gryglewski, R. J., Bunting, S., Moncada, S., Flower, R. J., and Vane, J. R. (1976) Prostaglandins 12,

6 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL 5. Sasaguri, Y., Morimatsu, M., Nakashima, T., Tokunaga, O., and Yagi, K. (1985) Biochem. Int. 11, Nishigaki, I., Hagihara, M., Maseki, M., Tomoda, Y., Nagayama, K., Nakashima, T., and Yagi, K. (1984) Biochem. Int. 8, Yagi, K., Inagaki, T., Sasaguri, Y., Nakano, R., and Nakashima, T. (1987) J. Clin. Biochem. Nutr. 3, Hirosumi, J., Ouchi, Y., Watanabe, M., Kusunoki, J., Nakamura, T., and Orimo, H. (1988) Biochem. Biophys. Res. Commun. 152, Sasaguri, Y., Kakita, N., Murahashi, N., Kato, S., Hiraoka, K., Morimatsu, M., and Yagi, K. (1993) Atherosclerosis 100, Arima, N., Sasaguri, Y., Ito, S., and Yagi, K. (1996) Biochem. Biophys. Res. Commun. 225, Yagi, K., Ishida, N., Komura, S., Ohishi, N., Kasai, M., and Kohno, M. (1992) Biochem. Biophys. Res. Commun. 183, Yagi, K., Kiuchi, K., Saito, Y., Miike, A., Kayahara, N., Tatano, T., and Ohishi, N. (1986) Biochem. Int. 12, Richter, C. (1993) FEBS Lett. 325,

A Simple Assay for Lipid Hydroperoxides in Serum or Plasma *

J. Clin. Biochem. Nutr., 20, 181-193, 1996 A Simple Assay for Lipid Hydroperoxides in Serum or Plasma * Kunio YAGI,1,** Sadaaki KOMURA,1 Norihiko KAYAHARA,2 Toshio TATANO,2 and Nobuko OHISHI1 1Institute