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Structural Differences in the Ability of Lysophospholipids to Inhibit Endothelium-Dependent Hyperpolarization by Acetylcholine in Rat Mesenteric Arteries
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
227, 479–483 (1996)
1532
Structural Differences in the Ability of Lysophospholipids to Inhibit Endothelium-Dependent Hyperpolarization by Acetylcholine in Rat Mesenteric Arteries Mitsuhiro Fukao,*,1 Yuichi Hattori,* Morio Kanno,* Ichiro Sakuma,† and Akira Kitabatake† Departments of *Pharmacology and †Cardiovascular Medicine, Hokkaido University, School of Medicine, Sapporo 060, Japan Received September 9, 1996 The effects of different lysophospholipids on endothelium-dependent hyperpolarization by acetylcholine were examined in rat mesenteric arteries. Lysophosphatidylcholine with 14 or longer carbon acyl chain significantly inhibited the hyperpolarization, while that with 12 or lesser carbon acyl chain was without effect. Lysophosphatidylcholine with unsaturated acyl chain also showed a potent inhibition. Lysophosphatidylinositol and lyso-platelet activating factor, but not phosphatidylcholine, lysophosphatidic acid, lysophosphatidylethanolamine or lysophosphatidylserine, suppressed the hyperpolarization. These results suggest that the length of the carbon acyl chain and the size of the polar head group may be crucial for the effects of lysophospholipids on endothelium-dependent hyperpolarization. Accumulation of these lysophospholipids may play an important role in endothelial dysfunction associated with atherosclerosis. q 1996 Academic Press, Inc.
Endothelium-derived hyperpolarizing factor (EDHF), which is distinct from nitric oxide or prostanoids, has been reported to play an important role in regulation of vascular tone especially at the level of the resistance vessels (1-3). EDHF is released from endothelial cells in response to vasoactive substances such as acetylcholine (ACh) and elicits vascular relaxation by opening of K/ channels in smooth muscle cells (4). The EDHF-mediated responses have been reported to be impaired in spontaneously hypertensive (5) and renal hypertensive rats (6). The pathological significance of the impaired EDHF-mediated responses is not established. However, the impairment of endothelium-dependent hyperpolarization may result in membrane depolarization, thus causing an increased vascular resistance. Elevated plasma level of low density lipoprotein (LDL) has been associated with the development of atherosclerosis (7). Oxidative modification of LDL has been implicated to be important for the atherogenic actions of LDL (8). Lysophosphatidylcholine (LPC), which is generated during oxidative modification of low density lipoproteins (9), appears to be a principal substance responsible for the impairment of endothelial function in atherosclerotic arteries (1012). We have previously reported that LPC inhibits the EDHF-mediated responses in rat mesenteric arteries (13). However, the EDHF-mediated responses may be also inhibited by lysophospholipids other than LPC, since different amphiphiles could elicit an inhibitory effect on endothelial function (14). In this study, we investigated the effects of a number of stractually 1 To whom correspondence should be addressed. Fax: /81-11-717-1139. Abbreviations used: EDHF, endothelium-derived hyperpolarizing factor; ACh, acetylcholine; LDL, low density lipoprotein; LPC, lysophosphatidylcholine; PC, phosphatidylcholine; LPA, lysophosphatidic acid; LPE, lysophosphatidylethanolamine; LPS, lysophosphatidylserine; LPI, lysophosphatidylinositol; lyso-PAF, lyso-platelet activating factor; G L-NNA, N -nitro-L-arginine.
479 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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different lysophospholipids on endothelium-dependent hyperpolarization by ACh in rat mesenteric artery. MATERIALS AND METHODS Electrophysiological experiments. Male Wistar rats (10-15 weeks old), weighing 230-340 g, were anesthetized with diethyl ether. The main trunk of the superior mesenteric artery was excised carefully and placed in a dish filled with oxygenated physiological salt solution at room temperature. The arteries were cleaned off adhering fats and connective tissues and cut into rings of 3-mm length. Care was taken to ensure that the endothelial layer was not damaged during the preparing procedure. When necessary, the endothelium was removed by gently rubbing the intimal surface with a moistened cotton ball. The artery was cut open along the longitudinal axis and pinned down on the bottom of an organ chamber (capacity 3 ml) with the endothelial side facing up. The preparation was superfused with warmed (37 7C) physiological salt solution aerated with 95% O2 and 5% CO2 at a constant flow rate of 7 ml/min. Physiological salt solution contained the followings (in mM); NaCl 118.2; KCl 4.7; CaCl2 2.5; MgCl2 1.2; KH2PO4 1.2; NaHCO3 25.0 and glucose 10.0. After an equilibration period of at least 60 min, glass microelectrodes filled with 3 M KCl (tip resistances 40 - 80 MV) were inserted into the smooth muscle cells from the intimal side. Membrane potentials were displayed continuously on an oscilloscope (Nihon-Kohden, VC- 10, Tokyo, Japan) and recorded on a chart recorder (Watanabe Sokki, WR3101, Tokyo, Japan). For data analysis, we used only the data of the experiments during which a single impalement was maintained. Drugs. The following compounds were used: ACh chloride, tetraethylammonium chloride and tetra-n-butylammonium bromide (Wako, Osaka, Japan); LPC (C18:0, C18:1, C16:0, C14:0, C12:0, C10:0, C8:0, C6:0), phosphatidylcholine (PC), lysophosphatidic acid (LPA), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidylinositol (LPI), lyso-platelet activating factor (lyso-PAF), NG-nitro-L-arginine (L-NNA), indomethacin and glibenclamide (Sigma Chemical, St. Louis, MO, USA). Lysophospholipids were dissolved in a mixture (1:1[vol/vol]) of methanol and chloroform and appropriate aliquots of the solution were dried with a stream of N2 gas and followed by sonication in distilled water. L-NNA was dissolved in 0.2 N HCl and indomethacin was prepared in 50 mM Tris. Glibenclamide was dissolved in 0.05 N NaOH. ACh was dissolved in distilled water. Further dilutions were made with physiological salt solution. Statistical analysis. All values are presented in terms of the mean { S.E.M. Statistical analysis of data was performed by Student’s t test. P values less than 0.05 were considered significant.
RESULTS
Resting membrane potentials of the mesenteric arterial smooth muscle cells were 051.9 { 0.3 mV (n Å 156). In the tissues with intact endothelium, ACh (1 mM) hyperpolarized the membrane potential by 016.7 { 0.2 mV (n Å 105). Removal of endothelium abolished the hyperpolarizing response to ACh (data not shown). ACh-induced hyperpolarization was not influenced by L-NNA (100 mM) or indomethacin (10 mM), and the hyperpolarization was eliminated by high K/ medium (25 mM), tetraethylammonium (10 mM) and tetrabutylammonium (500 mM), but not by glibenclamide (10 mM) (data not shown). The hyperpolarizing response to ACh was almost completely inhibited by LPC (C16:0; 10 mM) but not by LPC (C12:0) (Fig. 1A). As previously reported (13), the inhibition by LPC (C16:0) of Ach-induced hyperpolarization was concentration-dependent. The concentration of LPC (C16:0) required to inhibit the hyperpolarizing response to 1 mM Ach to the half was approximately 7 mM. The relationship between the acyl chain length and the inhibitory effect of LPC on ACh-induced hyperpolarization is shown in Fig. 1B. A significant inhibitory effect was observed that the carbon acyl chain length of LPC was 14 and longer. To further investigate structural characteristics for the inhibitory effect of LPC on ACh-induced hyperpolarization, we examined the effects of other phospholipids on ACh-induced hyperpolarization. LPC with unsaturated acyl chain (C18:1; 10 mM) also inhibited ACh-induced hyperpolarization (Fig. 2A). PC was without effect (Fig. 2A). Thus, only the lyso-type of phosphatidylcholine appears to express an inhibitory effect on ACh-induced hyperpolarization. Further study was performed to determine whether the 3* phosphate containing head region of glycerol backbone of lysophospholipids is important for the inhibitory effect on endothelium-dependent hyperpolarization. LPE (10 mM), LPS (10 mM) and LPA (10 mM) had no effect on ACh-induced hyperpolarization (Fig. 480
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FIG. 1. Effects of LPCs on the hyperpolarizing response to ACh. Panel A ; Actual tracings of the membrane potential changes by ACh. ACh was present for the periods indicated by the horizontal bars. 10 mM of LPC (C16:0) and LPC (C12:0) were added to the bath 20-30min before exposure to 1 mM of ACh. Each recording was obtained from the same preparation. Panel B ; Relationship between the acyl chain length and the inhibitory effect of LPC on ACh- induced hyperpolarization. After the control responses to 1 mM ACh had been obtained, 10 mM of LPCs with different acyl chain lengths were applied 20-30 min before the second challenge of ACh. The bars are shown as means { S.E.M. of 5-6 experiments. * Põ0.05, ***Põ0.001 vs. the corresponding control values.
2). However, LPI (10 mM) and lyso-PAF (10 mM) significantly suppressed ACh-induced hyperpolarization (Fig. 2B). DISCUSSION In the present study, structural characteristics for the ability of lysophospholipids to elicit an inhibitory effect on the EDHF-mediated response were explored. We measured ACh-induced endothelium-dependent hyperpolarization in rat mesenteric artery as a marker for EDHF release. The findings that hyperpolarization by ACh was not influenced by L-NNA, indomethacin or glibenclamide but eliminated by high K/ medium, tetraethylammonium and tetrabutylammonium, indicate that membrane hyperpolarization by ACh is mediated entirely by EDHF released from endothelial cells. LPC but not phosphatidylcholine elicited an inhibitory effect on ACh-induced hyperpolarization. This suggests that only the lyso-type of phospholipids could exert an inhibitory effect on the EDHF-mediated response. The mechanism by which LPC inhibit the EDHF-mediated response is not clear. Elevation of cytosolic Ca2/ in endothelial cells is a key step in the synthesis or release of EDHF (15). In cultured bovine aortic endothelial cells, LPC has been reported to inhibit phosphoinositide hydrolysis and the subsequent increase in cytosolic Ca2/ in endothelial cells (11). Furthermore, LPC is known to directly modulate ion channel function (16). The inhibitory effect of LPC on the EDHF-mediated response was dependent on the acyl chain length in the 1* carbon region of glycerol backbone of LPC. LPC with more than 14 carbon acyl chain inhibited the EDHF-mediated response. The dependency on the acyl chain length of LPC may be related to the access or insertion process of LPC to the membrane phospholipid bilayers. 481
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FIG. 2. Effects of different phospholipids on the hyperpolarizing response to ACh. After the control responses to 1 mM ACh had been obtained, 10 mM of phospholipids with different structures were applied 20-30min before the second challenge of ACh. The bars are shown as means { S.E.M. of 5-6 experiments. ***Põ0.001 vs. the corresponding control values.
Changes in the 3* phosphate containing head region of glycerol backbone also modulated the inhibitory effect of LPC on the EDHF-mediated response. The head group charge was not important for the inhibition of the EDHF-mediated response, because the effects of lysophospholipids varied regardless of whether they are negatively charged forms (LPI and LPS) or uncharged forms (LPC, LPE and lyso-PAF). The inhibitory effect of lysophospholipids may depend on a function of the size of the polar head. Lundbæk and Andersen (17) have shown 482
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that the potency of lysophospholipids to modulate gramicidin channel function is related to the size of the polar head group. Thus, lysophospholipids with large polar head group alter gramicidin channel function by altering the membrane deformation energy. In this experiment, the lysophospholipids with large polar head group, LPC, LPI and lyso-PAF, all inhibited the EDHF-mediated response, while those with small polar head group, LPS and LPE, were without effect. The present results suggest that lysophospholipids with long acyl chain and large polar head group elicit an inhibitory effect on the EDHF-mediated response. Lysophospholipids accumulate in pathological states such as atherosclerosis (9). Indeed, the concentration of LPC is increased manyfold in arteries from experimental atherosclerotic animals, which can be up to 11.2 mg/mg protein (18). Thus, the excessive concentration of LPC to lead to impairment of the EDHF-mediated response are present in atherosclerotic arterial lesions. The impairment of the EDHF-mediated response caused by lysophospholipids accumulation may play an important role in the development of vasomotor disturbance in atherosclerosis. ACKNOWLEDGMENTS We thank Miss S. Yamada for her secretarial assistance. This work was supported by a Grant-in-Aid 06770059 for Encouragement of Young Scientist from the Ministry of Education, Science and Culture of Japan and by the MITSUI Life Social Welfare Foundation.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Suzuki, H., Chen, G., and Yamamoto, Y. (1992) Jpn. Circ. J. 56, 170–173. Garland, C. J., Plane, F., Kemp, B. K., and Cock, T. M. (1995) Trends Pharmacol. Sci. 16, 23–30. Cohen, R. A., and Vanhoutte, P. M. (1995) Circulation 92, 3337–3349. Chen, G., Suzuki, H., and Weston, A. H. (1988) Br. J. Pharmacol. 95, 1165–1174. Fujii, K., Tominaga, M., Ohmori, S., Kobayashi, K., Koga, T., Takata, Y., and Fujishima, M. (1992) Circ. Res. 70, 660–669. Van De Voorde, Vanheel, J. B., and Leusen, I. (1992) Circ. Res. 70, 1–8. Kannel, W. B., Castelli, W. P., Gordon, T., and McNamara, P. M. (1971) Ann. Intern. Med. 74, 1–12. Steinberg, D., Parthasarathy, S., Carew, T. E., and Witztum, J. L. (1989) N. Engl. J. Med. 320, 915–924. Parthasarathy, S., Steinbrecher, U. P., Barnett, J., Witztum, J. L., and Steinberg, D. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3000–3004. Kugiyama, K., Kerns, S. A., Morrisett, J. D., Roberts, R., and Henry, P. D. (1990) Nature (Lond) 344, 160–162. Inoue, N., Hirata, K., Yamada, M., Hamamori, Y., Matsuda, Y., Akita, H., and Yokoyama, M. (1992) Circ. Res. 71, 1410–1421. Cox, D. A., and Cohen, M. L. (1996) Pharmacol. Rev. 48, 3–19. Fukao, M., Hattori, Y., Kanno, M., Sakuma, I., and Kitabatake, A. (1995) Br. J. Pharmacol. 116, 1541–1543. Mangin, E. L., Jr., Kugiyama, K., Nguy, J. H., Kerns, S. A., and Henry, P. D. (1993) Circ. Res. 72, 161–166. Chen, G., and Suzuki, H. (1990) J. Physiol. 421, 521–534. Eddlestone, G. T. (1995) Am. J. Physiol. 268, C181–190. Lundbæk, J. A., and Andersen, O. S. (1994) J. Gen. Physiol. 104, 645–673. Portman, O. W., and Alexander, M. (1969) J. Lipid Res. 10, 158–165.
483
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227, 479–483 (1996)
1532
Structural Differences in the Ability of Lysophospholipids to Inhibit Endothelium-Dependent Hyperpolarization by Acetylcholine in Rat Mesenteric Arteries Mitsuhiro Fukao,*,1 Yuichi Hattori,* Morio Kanno,* Ichiro Sakuma,† and Akira Kitabatake† Departments of *Pharmacology and †Cardiovascular Medicine, Hokkaido University, School of Medicine, Sapporo 060, Japan Received September 9, 1996 The effects of different lysophospholipids on endothelium-dependent hyperpolarization by acetylcholine were examined in rat mesenteric arteries. Lysophosphatidylcholine with 14 or longer carbon acyl chain significantly inhibited the hyperpolarization, while that with 12 or lesser carbon acyl chain was without effect. Lysophosphatidylcholine with unsaturated acyl chain also showed a potent inhibition. Lysophosphatidylinositol and lyso-platelet activating factor, but not phosphatidylcholine, lysophosphatidic acid, lysophosphatidylethanolamine or lysophosphatidylserine, suppressed the hyperpolarization. These results suggest that the length of the carbon acyl chain and the size of the polar head group may be crucial for the effects of lysophospholipids on endothelium-dependent hyperpolarization. Accumulation of these lysophospholipids may play an important role in endothelial dysfunction associated with atherosclerosis. q 1996 Academic Press, Inc.
Endothelium-derived hyperpolarizing factor (EDHF), which is distinct from nitric oxide or prostanoids, has been reported to play an important role in regulation of vascular tone especially at the level of the resistance vessels (1-3). EDHF is released from endothelial cells in response to vasoactive substances such as acetylcholine (ACh) and elicits vascular relaxation by opening of K/ channels in smooth muscle cells (4). The EDHF-mediated responses have been reported to be impaired in spontaneously hypertensive (5) and renal hypertensive rats (6). The pathological significance of the impaired EDHF-mediated responses is not established. However, the impairment of endothelium-dependent hyperpolarization may result in membrane depolarization, thus causing an increased vascular resistance. Elevated plasma level of low density lipoprotein (LDL) has been associated with the development of atherosclerosis (7). Oxidative modification of LDL has been implicated to be important for the atherogenic actions of LDL (8). Lysophosphatidylcholine (LPC), which is generated during oxidative modification of low density lipoproteins (9), appears to be a principal substance responsible for the impairment of endothelial function in atherosclerotic arteries (1012). We have previously reported that LPC inhibits the EDHF-mediated responses in rat mesenteric arteries (13). However, the EDHF-mediated responses may be also inhibited by lysophospholipids other than LPC, since different amphiphiles could elicit an inhibitory effect on endothelial function (14). In this study, we investigated the effects of a number of stractually 1 To whom correspondence should be addressed. Fax: /81-11-717-1139. Abbreviations used: EDHF, endothelium-derived hyperpolarizing factor; ACh, acetylcholine; LDL, low density lipoprotein; LPC, lysophosphatidylcholine; PC, phosphatidylcholine; LPA, lysophosphatidic acid; LPE, lysophosphatidylethanolamine; LPS, lysophosphatidylserine; LPI, lysophosphatidylinositol; lyso-PAF, lyso-platelet activating factor; G L-NNA, N -nitro-L-arginine.
479 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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different lysophospholipids on endothelium-dependent hyperpolarization by ACh in rat mesenteric artery. MATERIALS AND METHODS Electrophysiological experiments. Male Wistar rats (10-15 weeks old), weighing 230-340 g, were anesthetized with diethyl ether. The main trunk of the superior mesenteric artery was excised carefully and placed in a dish filled with oxygenated physiological salt solution at room temperature. The arteries were cleaned off adhering fats and connective tissues and cut into rings of 3-mm length. Care was taken to ensure that the endothelial layer was not damaged during the preparing procedure. When necessary, the endothelium was removed by gently rubbing the intimal surface with a moistened cotton ball. The artery was cut open along the longitudinal axis and pinned down on the bottom of an organ chamber (capacity 3 ml) with the endothelial side facing up. The preparation was superfused with warmed (37 7C) physiological salt solution aerated with 95% O2 and 5% CO2 at a constant flow rate of 7 ml/min. Physiological salt solution contained the followings (in mM); NaCl 118.2; KCl 4.7; CaCl2 2.5; MgCl2 1.2; KH2PO4 1.2; NaHCO3 25.0 and glucose 10.0. After an equilibration period of at least 60 min, glass microelectrodes filled with 3 M KCl (tip resistances 40 - 80 MV) were inserted into the smooth muscle cells from the intimal side. Membrane potentials were displayed continuously on an oscilloscope (Nihon-Kohden, VC- 10, Tokyo, Japan) and recorded on a chart recorder (Watanabe Sokki, WR3101, Tokyo, Japan). For data analysis, we used only the data of the experiments during which a single impalement was maintained. Drugs. The following compounds were used: ACh chloride, tetraethylammonium chloride and tetra-n-butylammonium bromide (Wako, Osaka, Japan); LPC (C18:0, C18:1, C16:0, C14:0, C12:0, C10:0, C8:0, C6:0), phosphatidylcholine (PC), lysophosphatidic acid (LPA), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidylinositol (LPI), lyso-platelet activating factor (lyso-PAF), NG-nitro-L-arginine (L-NNA), indomethacin and glibenclamide (Sigma Chemical, St. Louis, MO, USA). Lysophospholipids were dissolved in a mixture (1:1[vol/vol]) of methanol and chloroform and appropriate aliquots of the solution were dried with a stream of N2 gas and followed by sonication in distilled water. L-NNA was dissolved in 0.2 N HCl and indomethacin was prepared in 50 mM Tris. Glibenclamide was dissolved in 0.05 N NaOH. ACh was dissolved in distilled water. Further dilutions were made with physiological salt solution. Statistical analysis. All values are presented in terms of the mean { S.E.M. Statistical analysis of data was performed by Student’s t test. P values less than 0.05 were considered significant.
RESULTS
Resting membrane potentials of the mesenteric arterial smooth muscle cells were 051.9 { 0.3 mV (n Å 156). In the tissues with intact endothelium, ACh (1 mM) hyperpolarized the membrane potential by 016.7 { 0.2 mV (n Å 105). Removal of endothelium abolished the hyperpolarizing response to ACh (data not shown). ACh-induced hyperpolarization was not influenced by L-NNA (100 mM) or indomethacin (10 mM), and the hyperpolarization was eliminated by high K/ medium (25 mM), tetraethylammonium (10 mM) and tetrabutylammonium (500 mM), but not by glibenclamide (10 mM) (data not shown). The hyperpolarizing response to ACh was almost completely inhibited by LPC (C16:0; 10 mM) but not by LPC (C12:0) (Fig. 1A). As previously reported (13), the inhibition by LPC (C16:0) of Ach-induced hyperpolarization was concentration-dependent. The concentration of LPC (C16:0) required to inhibit the hyperpolarizing response to 1 mM Ach to the half was approximately 7 mM. The relationship between the acyl chain length and the inhibitory effect of LPC on ACh-induced hyperpolarization is shown in Fig. 1B. A significant inhibitory effect was observed that the carbon acyl chain length of LPC was 14 and longer. To further investigate structural characteristics for the inhibitory effect of LPC on ACh-induced hyperpolarization, we examined the effects of other phospholipids on ACh-induced hyperpolarization. LPC with unsaturated acyl chain (C18:1; 10 mM) also inhibited ACh-induced hyperpolarization (Fig. 2A). PC was without effect (Fig. 2A). Thus, only the lyso-type of phosphatidylcholine appears to express an inhibitory effect on ACh-induced hyperpolarization. Further study was performed to determine whether the 3* phosphate containing head region of glycerol backbone of lysophospholipids is important for the inhibitory effect on endothelium-dependent hyperpolarization. LPE (10 mM), LPS (10 mM) and LPA (10 mM) had no effect on ACh-induced hyperpolarization (Fig. 480
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FIG. 1. Effects of LPCs on the hyperpolarizing response to ACh. Panel A ; Actual tracings of the membrane potential changes by ACh. ACh was present for the periods indicated by the horizontal bars. 10 mM of LPC (C16:0) and LPC (C12:0) were added to the bath 20-30min before exposure to 1 mM of ACh. Each recording was obtained from the same preparation. Panel B ; Relationship between the acyl chain length and the inhibitory effect of LPC on ACh- induced hyperpolarization. After the control responses to 1 mM ACh had been obtained, 10 mM of LPCs with different acyl chain lengths were applied 20-30 min before the second challenge of ACh. The bars are shown as means { S.E.M. of 5-6 experiments. * Põ0.05, ***Põ0.001 vs. the corresponding control values.
2). However, LPI (10 mM) and lyso-PAF (10 mM) significantly suppressed ACh-induced hyperpolarization (Fig. 2B). DISCUSSION In the present study, structural characteristics for the ability of lysophospholipids to elicit an inhibitory effect on the EDHF-mediated response were explored. We measured ACh-induced endothelium-dependent hyperpolarization in rat mesenteric artery as a marker for EDHF release. The findings that hyperpolarization by ACh was not influenced by L-NNA, indomethacin or glibenclamide but eliminated by high K/ medium, tetraethylammonium and tetrabutylammonium, indicate that membrane hyperpolarization by ACh is mediated entirely by EDHF released from endothelial cells. LPC but not phosphatidylcholine elicited an inhibitory effect on ACh-induced hyperpolarization. This suggests that only the lyso-type of phospholipids could exert an inhibitory effect on the EDHF-mediated response. The mechanism by which LPC inhibit the EDHF-mediated response is not clear. Elevation of cytosolic Ca2/ in endothelial cells is a key step in the synthesis or release of EDHF (15). In cultured bovine aortic endothelial cells, LPC has been reported to inhibit phosphoinositide hydrolysis and the subsequent increase in cytosolic Ca2/ in endothelial cells (11). Furthermore, LPC is known to directly modulate ion channel function (16). The inhibitory effect of LPC on the EDHF-mediated response was dependent on the acyl chain length in the 1* carbon region of glycerol backbone of LPC. LPC with more than 14 carbon acyl chain inhibited the EDHF-mediated response. The dependency on the acyl chain length of LPC may be related to the access or insertion process of LPC to the membrane phospholipid bilayers. 481
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FIG. 2. Effects of different phospholipids on the hyperpolarizing response to ACh. After the control responses to 1 mM ACh had been obtained, 10 mM of phospholipids with different structures were applied 20-30min before the second challenge of ACh. The bars are shown as means { S.E.M. of 5-6 experiments. ***Põ0.001 vs. the corresponding control values.
Changes in the 3* phosphate containing head region of glycerol backbone also modulated the inhibitory effect of LPC on the EDHF-mediated response. The head group charge was not important for the inhibition of the EDHF-mediated response, because the effects of lysophospholipids varied regardless of whether they are negatively charged forms (LPI and LPS) or uncharged forms (LPC, LPE and lyso-PAF). The inhibitory effect of lysophospholipids may depend on a function of the size of the polar head. Lundbæk and Andersen (17) have shown 482
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that the potency of lysophospholipids to modulate gramicidin channel function is related to the size of the polar head group. Thus, lysophospholipids with large polar head group alter gramicidin channel function by altering the membrane deformation energy. In this experiment, the lysophospholipids with large polar head group, LPC, LPI and lyso-PAF, all inhibited the EDHF-mediated response, while those with small polar head group, LPS and LPE, were without effect. The present results suggest that lysophospholipids with long acyl chain and large polar head group elicit an inhibitory effect on the EDHF-mediated response. Lysophospholipids accumulate in pathological states such as atherosclerosis (9). Indeed, the concentration of LPC is increased manyfold in arteries from experimental atherosclerotic animals, which can be up to 11.2 mg/mg protein (18). Thus, the excessive concentration of LPC to lead to impairment of the EDHF-mediated response are present in atherosclerotic arterial lesions. The impairment of the EDHF-mediated response caused by lysophospholipids accumulation may play an important role in the development of vasomotor disturbance in atherosclerosis. ACKNOWLEDGMENTS We thank Miss S. Yamada for her secretarial assistance. This work was supported by a Grant-in-Aid 06770059 for Encouragement of Young Scientist from the Ministry of Education, Science and Culture of Japan and by the MITSUI Life Social Welfare Foundation.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
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