AL0671, A new potassium channel opener, inhibits nonenzymatic glycation of protein and LDL oxidation

Gen. Pharmac. Vol. 27, No. 2, pp. 257-262, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA.

ISSN 0306-3623/96 $15.00 + .00 0306-3623(95)O0O97-6 All rights reserved ELSEVIER

AL0671, A New Potassium Channel Opener, Inhibits Nonenzymatic Glycation of Protein and LDL Oxidation Takeshi Yamauchi*, Sumio Matzno, Teruaki lmacla, Masahiro Eda, Yoshihisa Inoue and Norifumi Nakamura BASIC RESEARCH LASORAVOIUES,R ~ a c n DIwsloN, TaE GaL,~ Caoss Coaroaa~oN. 2-25-1, SnODAI-OHTANI,HIRAKATA,OSAKA573, JAPANTEL: 81-720-56-9204; FAX: 81-720-57-5020

ABSTRACT. 1. T h e effects ofALO671, a novel potassium channel opener, on protein glycation and low-density lipoprotein (LDL) oxidation were tested. 2. AL0671 dose-dependentlyinhibitedbothfluorescencedeveblnnentofbovit~.eserumalbuminand cross.linking of lysoryme. These inhibitory e ffec~ for glycation we re no less potent than amln ~ . 3. AL0671 dose-dependently inhibited both increase in negative charge and aim B- 100 fragmentation during incubation of LDL with Cu z+. In addition, AL0671 signgficantly decreaJa~d the LDL degradation in rat peritoneal macrophages. 4. Neither pinacidil nor levcromakalim inhibited protein glycation and LDL oxidation. 5. Antioxidant properties of AL0671 might be due to its potent electron.donating ability, and this agent is expected to be useful for hypertensive diabetes, oEs px~,mo,c 27;2:257-262, 1996. KEY WORDS. Potassium channel opener, AL0671, glycation, oxidation INTRODUCTION It has been pointed out that hyperglycemia-induced formation of advanced glycation end products (AGE) plays a central role in the pathogenesis of diabetic complications. AGE accumulation causes dysfunctional change in extracellular matrix (e.g., collagen), abnormal receptor-mediated production of cytokines (e.g., interleukin-1, tumor necrosis factor, insulin-like growth factor, and platelet-derived growth factor), quenching of the endothelium-derived relaxing factor, and altered function of intracellular proteins such as basic fibroblast growth factor (Brownlee, 1994). Elevated plasma concentrations of low-density lipoprotein (LDL) are associated with acceleration ofatherogenesis. Studies both in vitro and in vivo support the concept that the uptake of oxidized LDLby macrophage through a specific receptor, a scavenger receptor, develops the fatty streak, the earliest lesion in atherogenesis (Steinberg et al., 1989). Aminoguanidine is a nucleophilic hydrazine compound that prevents AGE formation by reacting with active dicarbonyl intermediates such as 3-deoxyglucosone (Brownlee et al., 1986; Edelstein and Brownlee, •992). The effectiveness of aminoguanidine in diabetic pathology has been investigated in nerve (Yagihashi eta/., 1992), retina (Hammes et al., 1991), and kidney (Soulis-Liparota et al., 1991), suggesting that pharmacological inhibition of AGE is able to prevent diabetic complications. Recent studies revealed that aminogu anidine also possesses inhibitory activity against oxidative modification ofLDL (Picard et al., 1992; Requena et al., 1992). AL0671 is a newly synthesized potassium channel opener derivative ofcyanoguanidine with a 3-pyridilgroup ((+)-N-(6-amino-3-pyridil).N'[ 1S,2R,4R)-bicyclo[2.2.1 ]hept-2yl]-N'cyanoguanidine hydrochloride, AL0670" HCI), that activates ATP-sensitive potassium channel (Matzno et al., 1995) and produces sustained decrease in blood pressure (Eda et al., 1994). In addition to its vasodilating activities, AL0671 significantly lowered plasma triglycerides of hyperlipidemic Zucker rats through activation of both lipoprotein lipase and hepatic triglyceride lipase (Matzno et al., 1994). *To whom all correspondence should be addressed. Received 2 May 1995.

Based on a structural similarity between aminoguanidine and the cyanoguanidil moiety of AL0671 (Fig. 1), we tested whether AL0671 inhibits nonenzymatic glycation of proteins and LDL oxidation in in vitro studies. MATERIALS A N D M E T H O D S Mater/a/s AL0671, pinacidil, and levcromakalim were synthesized in the Basic Research Laboratories, Research Division, The Green Cross Corpora. tion. Other materials were obtained as follows: Human LDL from Chemicon (Temecula, CA), bovine serum albumin (BSA) from Sigma Chem. (St. Louis, MA), chicken egg white lysozyme from Biozyme Laboratories Ltd., aminoguanidine hydrochloride from Wako Chemicals (Osaka, Japan), 125-iodine sodium salt from NEN Research Products, and aspirin and iodine monochloride from Nacalai Tesque Inc. (Kyoto, Japan). AL0671 was derived from AL0670 by dissolving it in equivalent hydrochloric acid. Other drugs were dissolved in dimethylsulfoxide. Each drug solution was stored at - 2 0 ° C until use. Glycation

o[ protein

BSA (50 mg/ml) or lysozyme (10 mg/ml) was mixed with 250 mM of D-glucose and 0.5 to 10 mM of drug in 0.5 M sodium phosphate buffer, pH 7.4 containing 0.1 mg/ml pepstatin, 0.5 ltg/ml leupeptin, 100 IU/ ml penicillin, 0.1 mg/ml streptomycin, 1 mM EDTA and 3 mM sodium azide. After filtration through a sterile 0.22 gm filter, the mixture was put into a light-tight, sealed tube and kept for 3 to 4 weeks at 37°C. Aliquots were removed and assayed for AGE formation by the measurements of fluorescence and cross-linked oligomer proteins. Fluorescence of the BSA mixture was measured by the fluorometer (Fluoroscan II: Titertek) at 460 nm upon excitation at 355 nm and difference of the fluorescence (AF) before and after the incubation was calculated (Suarez et al., 1989). To avoid artifacts originated from the fluorescence of a drug per se, fluorescence development was calculated by subtracting AF of the reaction mixture with AF of the reaction mixture that lacks glucose. Inhibition of the fluorescence development by a

T. Yamauchi et al.

258 cyanoguanidyl

~ H2N

I~I~Nc~'~t[ HCI

H2N'/I~ly NH2

o~"100, 'E 80.

8"

1

• 0.5 mM [] 2.5 mM • 5 mM [] 10 mM

../

/

60,

NH

•

bmin°guanidine~

F I G U R E 1. ChemicalstructuresofALO671andaminoguanidine.

drug (%) was calculated by dividing the fluorescence development of a mixture containing the drug with the fluorescence development of a mixture free from the drug. In the cross-linking experiment lysozyme, rather than BSA was preferred, because multimers of lysozyme make distinct bands on SDSpolyacrylamidegel electroph0resis(SDS-PAGE) (Shin eta/., 1988). Aliquot of the incubation mixture was applied to a 4% to 20% gradient gel.

Chemical modil~cation and uSI.labeling o f L D L Acetyl-LDL was prepared by a reaction of LDL with acetic anhydride (Goldstein et al., 1979). Native LDL and acetyl-LDL were radioiodinated with NalZSIin a glycine-NaOH buffer, pH 10.0 using the iodine monochloride method (Bilheimer et al., 1972). gadiolabeled LDL was then dialyzed against phosphate-buffered saline (PBS) containing 0.5 mM EDTA for 17 hr and, subsequently, against PBS without EDTA for 48 hr. The final concentrations are 250 gg/ml for IzSI-acetyl-LDL. Labeled lipoproteins were stored at 4°C and used within a week.

Oxidation and characterization o f L D L LDL oxidation was initiated by adding copper sulfate (CuSO4) described by Hoff et al., (1992). To test the effect of drugs on LDL oxidation, 1zSI-LDL (100 mg/ml) was incubated with 10 ~tM of CuSO4 in the presence or absence of the drug (0.1 to 50 mM) in PBS at 37°C for 24 hr. The reaction was terminated by adding 0.1 MM of EDTA. Aliquot was subjected to SDS-PAGE and agarose gel electrophoresis. SDSPAGE was carried out on 2% to 15% gradient gel. The gel was subjected to autoradiography using Fuji RX film (Fuji Tokyo, Japan) after fixation with 25% methanol and 10% acetic acid and dehydrated. Agarose gel electrophoresis was performed at pH 8.6 in 0.06 M barbital buffer on mixed gel of 0.4% agarose and 0.12% agar. Following electrophoresis, the gel was fixed with 25% methanol, 10% acetic acid, dehydrated, and autoradiographed. In addition, aliquot of the incubation mixture were provided for the degradation assay.

Preparation of rat peritoneal macrophages Resident rat peritoneal macrophages were obtained from male Wistar rats (Rattus norvegicus, weighing 250 to 300 g) by lavage with PBS according to the method of Quinn et al. (1985). After centrifugation at 150 g for 10 min at room temperature, peritoneal cells were suspended in Dulbecco's modification Eagle's medium (DMEM) containing 10% fetal calf serum, 100 IU/ml penicillin, and 0.1 mg/ml streptomycin and plated at 2x 106 cells/ml/well into 12-wells tissue culture plates (Sumitomo Bakelite Co. Ltd., Tokyo, Japan). After 24 hr of incubation, plates were washed tw/ce with PBS for removal of non-adherent cells and the adherent macrophages were used in the following degradation experiments.

4o. g

"6 20. g ~¢¢-

o LEVCRO PINACIDIL MAKALIM

AL0671

ASPIRIN

AMINOGUANIDINE

F I G U R E 2. Effects of potassium channel openers, aspirin, and aminoguanidine on the fluorescence development of bovine serum albumin (BSA) during incubation with glucose. BSA (50 mg/ml) were incubated with 250 m M of glucose and drugs at 37°C for 4 weeks and then the fluorescence was measured. Inhibition of the fluorescence development was calculated as described in "Materials and Methods." All values represent mean_+S.E. (n =4). Significantly different from 0.5 m M each drug; *p
Degradation assay Oxidized 1251-LDLwas diluted to 1 p.g/ml with DMEM containing 0.1% BSA, 100 IU/ml penicillin, and 0.1 mg/ml streptomycin. Aliquot (1 ml) was added into adherent macrophages of cell-free wells. After 4-hr incubation, 200 I11 of the medium was removed, mixed with 100 gl of 20% trichloroacetic acid, and stored at 4 °C for 30 rain. After centrifugation, the I25Iradioactivity in the supernatant was measured (Hoff eta[., 1992). At the end of the experiments, the cells were washed twice with ice-cold PBS and dissolved in 0.5 M NaOH. The content of cell protein was determined by Bio-Rad Protein Assay with human immunoglobulin as a standard.

Statistical analysis Statistical significance was assessed by one-way ANOVA, then Dunnett's multiple comparison method. Data are presented as mean_+S.E. RESULTS

Inhibition o[ protein glycation by AL0671 We evaluated the inhibitory effect of AL0671 against AGE formation, by measuring its effects on fluorescence development and cross-linked protein formation during incubation with protein and glucose. Fluorescence generation in a mixture of BSA and glucose after 4 weeks of incubation was inhibited by AL0671 in a dose-dependent manner (Fig. 2). At 10, 5, 2.5, and 0.5 raM, AL0671 inhibited fluorescence development by 52%, 32%, 23%, and 12%, respectively. The inhibitory effect was comparable to that of aspirin. Levcromakalim and pinacidil did not show any detectable inhibition at the tested concentrations. Generation of cross-linked lysozyme oligomers (i.e., dimer, trimer, and tetramer) was detectable by SDS-PAGE during incubation with glucose for 3 to 4 weeks (lanes 6 and 12, Fig. 3). AL0671 treatment markedly reduced intensities of the oligomer bands. Judged from the intensities of oligomer bands (lanes 3 and 9), the inhibitory effect of AL0671 was apparently stronger than that of aspirin (lanes 4 and

AL0671 on Glycation and Oxidation

259 that AL0671 prevented the oxidation of LDL, but not the following step of its degradation by macrophage. LDL oxidized in the presence of levcromakalim or pinacidil were almost completely degraded.

Mw

1 2 3 4 5 6 7 8 9101112std.

106 kD 80 kD Tetramer - * Trimer Dimer --* Monomer --*

49.5 kD 32.5 kD 27.5 kD 18.5 kD

FIGURE 3. SDS-PAGE of glycated lysozyme. Lysozyme (10 mg/ ml) were incubated with 250 mM of glucose and 10 mM of drags at 37°C for 3 (lane 1-6) or 4 (lane 7-12) weeks and subjected to SDS-PAGE using 4%-20% gradient gel. Gel was stained with Coomassie blue. Drugs used were levcromakalim (lane 1, 7), pinaci. dil (lane 2, 8), ALO671 (lane 3, 9), aspirin (lane 4, 10), and aminoguanidine (lane 5, 11). No drug was included in the samples of lanes 6 and 12.

10) and seemed to be comparable to that of aminoguanidine. Neither levcromakalim (lanes 1 and 7) nor pinacidil (lanes 2 and 8) had detectable changes on cross-linked lysozyme formation. Inhibition

o f LDL

To examine whether or not AL0671 can inhibit oxidative modification of LDL, we incubated LDL with Cu 2+ in the presence of AL0671 and measured the mobility on agarose gel electrophoresis. Incubation of LDL with Cu 2+ increased its electrophoretic mobility (lane 4, Fig. 4). This result indicates that the net negative charge increased during oxidation, because addition of AL0671 to the pre-oxidi2ed LDL did not affect its electrophoretic mobility (data not shown). Inclusion of AL0671 in the LDL oxidation step, however, prohibited the negative charge increment (lanes 10, 11). With 10 mM AL0671, modified LDL migrated to the position almost indistinguishable from that of native LDL (lane 10). Treatment with I mM AL0671 significantly improved the mobility shift (lane 11), whereas 0.1 mM AL0671 was not effective (lane 12). Aminoguanidine also dose-dependently corrected the increased mobility shift (lanes 6-8); however, it was less potent than AL0671. Pinacidil and levcromakalim did not protect LDL from the oxidative modification within the tested concentrations (lanes 13-17). Antioxidant properties of AL0671 were further evaluated by SDSPAGE (Fig. 5). Intact apolipoprotein 13-100 (apoB-100) band of native LDL (lane 1) was degraded by Cu E+-mediated oxidation (lane 2). AL0671 (1 to l0 raM) protected apoB-100 from oxidation (lanes 9 and 10). In contrast, the apoB band was completely fragmented when LDL was oxidized in the presence of 1 to 50 mM of aminoguanidine (lanes 4-8). Neither pinacidil (lanes 12-14) or levcromakalim (lanes 15, 16) protected apoB fragmentation from ox!dation (lane 5). Because oxidized LDL is effectively taken up and degraded by macrophage through binding to scavenger receptors, we oxidized LDL with Cu E+ in the presence of AL0671 and measured its degradation by macrophage. When Cu2+-oxidized LDL or acetyl-LDL was incubated with macrophage for 4 hr, a more than 10-fold increase in degradation was observed compared with untreated LDL (Fig 6.). Addition of 1 or 5 mM AL0671 in the oxidation mixture almost completely inhibited the degradation. On the other hand, after LDL was oxidized, its de gradation was not inhibited by AL0671 (data not shown). These findings indicate

DISCUSSION In the present study, we demonstrated that AL0671 significantly prevents nonenzymatic protein glycation and LDL oxidation. From the dual inhibitions of AL0671, we assumed that AL0671 affects to the common modification step, the free radical formation induced by metal cations (such as Cu 2+ and re3+), in the generation of glycation and oxidation as summarized in Fig 7. Glycation of proteins involves a complex series of reactions, including initial attachment of glucose to protein by Schiffbase formation followed by Amadori rearrangement to generate stable ketoamine. Further steps from ketoamine to yield AGE are poorly characterized; however, peroxide radicals and hydroxyl radicals are produced from ketoamine group in the presence of transition metals (Kawakishi et al., 1991). Autoxidation of glucose also yields H202 and free radical intermediates in a metal-catalyzed process (Wolffand Dean, 1987); therefore, antioxidants, metal-chelates, and radical scavengers have been shown to prevent the florescence development and cross-linking of proteins. Those are the indicators of the AGE formation during incubation with glucose (Fuet al., 1994; Khatami et al., 1988). At the initial step of LDL oxidation, a small amount of lipid hydroperoxide in the LDL particle is degraded by the endothelial cells (EC) and lipid peroxyl radical and lipid alcoxyl radical are generated. Steinbrecher et al. (1984) have demonstrated that the existence of transition metals (probably Cu 2÷) are crucial for EC-modified LDL. There follows a dramatic increase of the number of free radicals by the radical chain reactions leading to fragmentation of the fatty acid chains. After that, the fragments of the peroxidized fatty acids attach to lysine e-amino group of apoB- 100, and apoB- 100 is fragmented (Steinbrecher, 1987a; Steinbrecher et al., 1987b). The fragmented apoB- 100 are not recognized by the LDL receptor in the liver; this plays a central role in LDL clearance and lipid homeostasis, and results in the accumulation of oxidized LDL in the circulation (Steinberg et al., 1989). On the other hand, LDL particles become negatively charged as a result of oxidation.

1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17

FIGURE 4. Agarose gel electrophoresis of Cu z+-oxidized LDL in the presence of aminoguanidine and potassium channel openers. p2SI]LDL was incubated with 10 8M of CuSO4 and drugs for 24 hr at 37°C. T h e reaction was terminated by adding O. 1 mM EDTA, then applied to the agarose gel. Lane 1: intact LDL; 2: acetybLDL; 3: without CuSO4, 4:10 8M CuSO4; 5-9: aminoguanidine (50, 25, 10, 5, 1, mM); 10-12: AL0671, (10, 1, 0.1 mM); 13-15: pinacidil (10, 1, O.1 mM); 16 and 17: levcromakalim (1, 0.1 mM).

260

T. Yamauchi eta/.

Mw std. 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6

ApoB-100 200 kD---97 kD--" 69 k D ~ 46 kD--*

FIGURE 5. SDS-PAGE of Cu :+. oxidized LDL in the presence of aminoguanidine and potassium channel openers. The same sam. pies applied to agarose gel (Fig. 4) were analyzed by SDS-PAGE using 2%- 15% gradient gel Lane 1: intact LDL; 2:10 m M CuSO4; 3: without CuSO,,; 4-8: aminoguani. dine (50, 25, 10, 5, 1 mlVO; 9-11: AL0671 (10, 1, 0.1 mM); 12-14: pinacidil (10, 1, 0.1 mM); 15, 16: levcromakalim (1, 0.1 raM).

30 kD---*

These oxidized LDL particles are more rapidly incorporated by macrophage through the scavenger receptor (Kodama et al., 1990) and/or high-affinity Fc receptors for IgG (Bigler eta/., 1990; Stanton eta/., 1992). Incidentally, how does AL0671 affect the free radical formation induced by metal cations? We focused on the structural feature of AL0671 itself, especially the 6-amino unit of AL0671. In our calculation, HOMO energy level of 6-aminopyridine of AL067I is -0.29819 eV, whereas that of the pyridine unit alone if -0.35593 eV, indicating that the 6-amino unit of AL0671 increases the electron-donating ability of this molecule. This electron donation is independent on its potassium channel opening action; in fact, other potassium channel openers, pinacidil (cyanoguanidine derivative) and levcromakalim (benzopyrane derivative), could not inhibit protein glycation (Figs. 2 and 3) and LDL oxidation (Figs. 4, 5, and 6). Figure 7 shows the possible inhibitory mechanism of AL0671 on protein glycation and LDL oxidation. AL0671 might inhibit the metal cation (probably Cu2+)-induced peroxide radical formation by electron-donation to the radicals, resulting in

the termination of subsequent AGE formation and the radical chain reaction in LDL particles (including apoB-100 fragmentation). AL0671 inhibited the oxidative fragmentation of apoB-100 (Fig. 5), suggesting that AL0671 is expected to maintain normal LDL metabolism. In contrast, aminoguanidine failed to protect apoB-100, even at the concentrations that prevent the increment of negative charge (Fig. 4), as described previously (Scaccini et al., 1994). Although further studies are necessary to evaluate its in vivo effects, the observed properties ofAL0671 may benefit treatment of hypertension associated with diabetes, because hyperglycemia-induced protein glycation and lipid peroxidation are enhanced in diabetic subjects (Lyons, 1992; Monnier eta/., 1986). In this study, we used BSA and lysozyme for observation of AGE formation; however, a recent study indicated that LDL is also glycated in vivo and glycated-LDL is also incorporated rapidly into macrophage (Lopes-Virella et al., 1988). Furthermore, Maggi eta/. (1993) described LDL from hypertensive patients as being more sensitive to oxidation. In conclusion, AL0671 is an antihypertensive agent with many additional benefits, such as hypotriglycemic activity, antioxidative activity, and inhibition of protein glycation.

4

CONCLUSION

E ~a

AL067 I, an antihypertensive agent, possesses many additional benefits, such as hypotriglycemic activity, antioxidative activity, and inhibition of protein glycation.

.J

9

SUMMARY • AL0671 • Levcromakalim • Pinacidil ~ ";" ~"0.11 5 1 110 -= ~ -= ~0 ~" ¢~ L'-Drugs(mM)~

FIGURE 6. Anti-oxidative effects of potassium channel openers on LDL degradation in rat peritoneal macrophages. LDL (100 pgl ml) was preincubated with 10 pM of CuSO4 and drugs at 37°C for 4 hr. Then the LDL (1 pg) was added to the I ml of medium and incubated with macrophages. Degradation of LDL was measured from trichloroacetic acid-soluble radioactivity liberated into culture medium. Drugs used were AL0671 (E]), levcromakalim (0) and pinacidil (D). All values represent mean_+ S.E. (n = 3). Significantly different from CuZ+(+ ) group; *p<0.05, **p<0.O1.

The accumulating data suggest that advanced glycation end products (AGE) formation would play a central role in the pathogenesis of diabetic complications. Oxidative stress is also enhanced in diabetes, and may increase the risk of atherogenesis. AL0671, a novel potassium channel opener of cyanoguanidine derivative with a 3-pyridil group, is a potent long-lasting antihypertensive agent with serum triglyceridelowering effects. In the present study, the effects of AL0671 on protein glycation and LDL oxidation were studied based on its structural similarity with aminoguanidine. AL0671 (0.1 to 10 raM) dose-dependently inhibited both fluorescence development of bovine serum albumin and cross-linking oflys0zyme when incubated with glucose. These inhibitory actions of AL0671 on AGE formation were comparable to those of aminoguanidine. In addition, AL0671 (0.1 to 5 raM) dose-dependently inhibited oxidative modifications of L D L by Cu 2÷ as evidenced by lack

AL0671 on Olycation and Oxidation

261 Pro,°,,',

Reducing sugar

- NH2 ~ .H20

Protein

~,

"',,,

~ H

~

(~H:~ (~'"O

(?HOH)4

(~HOH)s

CH20 H

".

CH2OH

Schiff base

Amadori Product

" ~ Ape B-100 ~ k ~ - - -

Free cholesterol, Phospholipid coat

0 2

A'H202--D..-.OH 0 2-

~'~-Cholesteryl ester, Tdglycedde c o r e

~

L O O H + Cu 2+ --* L e o - + Cu + + H + A - N = N - A ~ 2A, + N 2 L O O H + Cu + --" LO. + Cu 2+ + -OH 1~ A, + O 2 --" AO2" I

LH

...................... ::::--:::::~/I ~

L

~'~LO2~'t

P C O O H .................... . ~

/L~

LH

Increment of negative charge Fragmentation of ApoB-100 and Upids

F I G U R E 7. Possible i n h i b i t o r y m e c h a n i s m of A L 0 6 7 1 in the process of protein glycation and LDL oxidation.

of increase in negative charge, apoB-100 fragmentation, and degradation by rat peritoneal macrophage, whereas aminoguanidine did not prevent apoB-100 fragmentation. Other potassium channel openers, pinacidil and levcromakalim, neither inhibited A G E formation nor LDL oxidation. The potential effects of AL0671 on protein glycation and LDL oxidation may be beneficial in treatment of hypertension associated with diabetes.

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Hoff H. F., Whitaker T. E. and O'Neil J. (1992)Oxidation of low density lipoprotein leads to particle aggregation and altered macrophage recognition. ]. Biol. Chem. 267, 602-609. Kawakishi S., Tsunehiro J. and Uchida K. (1991) Autooxidative degradation of Amadori compounds in the presence of copper ion. Carboh3dr. Res. 211, 167-171. Khatami M., Suldan Z., David I., Li W. and Rockey J. H. (1988) Inhibitory effects of pyridoxal phosphate, ascorbate and aminoguanidine on nonenzymatic glycosylation. Life Science 43, 1725-1731. Kodama T., Freeman M., Rohrer L., Zabrecky J., Matsudaira P. and Krieger M. (1990) Type I macrophage scavenger receptor contains a-helical and collagen-like coiled coils. Nature 343, 531-535. Lopes-Vireila M. F., Klein R. L., Lyons T. J., Stevenson H. C. and Witztum J. L. (1988) Glycosylation of low-density lipoprotein enhances cholesteryl ester synthesis in human monocyte-derived macrophages. Diabetes 37, 550-557. Lyons T. J. (1992) Lipoprotein glycation and its metabolic consequence. D/abetes 41(suppl. 2), 67-73. Maggi E., Marchesi E., Ravetta V., Falaschi F., Finardi G. and Bellomo G. (1993) Low-density lipoprotein oxidation in essential hypertension. ]. Hypertens. 11, 1103-1111. Matzno S., Gohda M., Eda M., Ebisu H., Uno S., Ishida N., Nakamura N. and Yamanouchi K. (1994) A possible mechanism of action of a new potassium channel opener, AL0671, on lipid metabolism in obese Zucker rats. ]. Phannac. exp. Ther. 271, 1666-1671. Matzno S., Sato R., Takai H., Aida Y., Karasaki S., Oyaizu M., Nakamura N. and Katori R. (1995)The effect of AL0671, a novel potassium channel opener, on potassium current in rat aortic smooth muscle cells. Gen. Pharmac. in press. Monnier V. M., Vishwanath V., Frank K. E., Elmets C. A., Dauchot P. and Kohn R. R. (1986) Relation between complications of type I diabetes mellitus and collagen-linked fluorescence. New Engl. J. Med. 314, 403-408. Picard S., Parthasarathy S., Fruebis J. and Witztum J. L. (1992) Aminoguanidine inhibits oxidative modification of low density lipoprotein and the subsequent increase in uptake by macrophage scavenger receptors. Proc. Natl. Acad. Sci. USA 89, 6876-6880. Quinn M. T., Parthasarathy S. and Steinberg D. (1985) Endothelial cell-derived chemotactic activity for mouse peritoneal macrophages and the effects of modified forms of low density lipoprotein. Proc. natl. Acad. Sci. USA 82, 5949-5953.

262 Requena J. R., Vidal P. and Caezas-Cerrato J. (1992) Aminoguanidine inhibits the modification of proteins by lipid peroxidation derived aldehydes: a possible antiatherogenic agent. Diabetes Res. 20, 4349. Scaccini C., Chiesa G. and Jialal L (1994) A critical assessment of the effects of aminoguanidine and ascorbate on the oxidative modification of LDL: evidence for interference with some assays of lipoprotein by aminoguanidine. J. Lipid Res. 35, 1085-1092. Shin D. B., Hayase F. and Kato H. (1988) Polymerization of proteins caused by reaction with sugars and the formation of 3-deoxyglucosone under physiological conditions. Agr/c. biol. Chem. 52, 1451-1458. Soulis-Liparota T., Cooper M., Papazoglou D., Clarke B. and Jerums G. (1991) Retardation by aminoguanidine of development of albuminuria, mesangial expansion, and tissue fluorescence in streptozotocin-induced diabetic rat. Diabetes 40, 1328-1334. Stanton L. W., White R. T., Bryant C. M., Protter A. A. and Endemann G. (1992) A macrophage Fc receptor for IgG is also a receptor for oxidized low density lipoprotein. ]. biol. Chem. 267, 22446-22451. Steinberg D., Parthasarathy S., Carew T. E., Khoo J. C. and Witztum J. L. (1989) Beyond cholesterol: Modifications of low-density lipoprotein that increase its atherogenicity. New Engl. ]. Med. 320, 915-924.

T. Yamauchi et al. Steinbrecher U. P. (1987a) Oxidation of human low density lipoprotein results in derivatization of lysine residues of apolipoprotein B by lipid peroxide decomposition products. ]. biol. Chem. 262, 3603-3608. Steinbrecher U. P. and Witztum J. L. (1984) Glycosylation of low-density lipoproteins to an extent comparable to that seen in diabetes slows their catabolism. Diabetes 33, 130-134. Steinbrecher U. P., Witztum J. L., Parthasarathy S. and Steinberg D. (1987b) Decrease in reactive aminogroups during oxidation or endothelial modification of LDL: correlation with changes in receptor-mediated catabolism. Arteriosderosis 1, 135-143. Suarez G., Rajaram R., Oronsky A. L. and Grawinowicz M. A. (1989) Nonenzymatic glycation of bovine serum albumin by fructose (fructation). Comparison with the Maillard reaction initiated by glucose. J. biol. Chem. 264, 36743679. WolffS. P. and Dean R. T. (1987) Glucose autoxidation and protein modification. The potential role of'autoxidative glycosylation' in diabetes. Biochem. J. 245, 243-250. Yagihashi S., Kamijo M., Baba M., Yagihashi N. and Nagai K. (1992) Effect of aminoguanidine on functional and structural abnormalities in peripheral nerve of STZ-induced diabetic rats. Diabetes 41, 47-52.