Materials and Methods

1 Supplement to Role of Glutaredoxin-Mediated Protein S-Glutathionylation in Cellular Nitroglycerin Tolerance

2 Pei-Suen Tsou, Vamsi Addanki, Jessica Haas, Nathaniel A. Page and Ho-Leung Fung.

3 1Materials and Methods 2 3Materials. The sources of various materials and reagents are listed as follows: LLC-PK1 cells

4(American Type Culture Collection, Rockville, MD); Ham’s F-12 medium, Dulbecco’s Modified

5Eagle Medium without L-methionine and L-cysteine, Opti-MEM® I reduced serum medium,

6fetal bovine serum (FBS), 104 U/ml penicillin, 10 mg/ml streptomycin, Lipofectamine 2000™,

7pcDNA3.1(+) expression vector, Lipofectamine™, and nitrocellulose membrane (Invitrogen,

8Carlsbad, CA); NTG solution (30% propylene glycol, 30% ethanol; Abbott Laboratories, North

9Chicago, IL); NitroQuick sublingual NTG tablets (0.6 mg, Ethex Corporation. St. Louis, MO);

10Alzet osmotic pumps (DURECT Corporation, Cupertino, CA); the ECL-enhanced

11chemiluminescence system (Pierce, Rockford, IL); creatine kinase (CK) assay medium cocktail

12(Stanbio Laboratory, Boeme, TA); L-35S-cysteine, and anti-mouse IgG HRP-linked conjugates

13(Amersham Biosciences Corp., Piscataway, NJ); anti-S-glutathionylated protein antibodies

14(Virogen, Watertown, MA); anti-GRX-1 polyclonal antibody (recombinant human protein, Lab

15Frontier, Seoul, Korea); cyclic GMP enzyme immunoassay kit (Assay Designs, Ann Arbor, MI);

16and all other chemicals (Sigma, St. Louis, MO). The transfection vector, pcDNA3-His-GRX, was

17kindly provided by Dr. Yong J. Lee (University of Pittsburgh) and NTG in 5% dextrose solution

18was kindly donated by Schwarz Pharma (Monheim, Germany).

19 4 Supplement to Role of Glutaredoxin-Mediated Protein S-Glutathionylation in Cellular Nitroglycerin Tolerance

6 20Cell culture. LLC-PK1 cells were cultured in Ham’s F-12 medium, containing 15% FBS, 100

21U/ml penicillin, and 100 g/ml streptomycin. They were maintained in a humidified incubator at

o 2237 C and 5% CO2. Cells between passage 6 and 16 were used in all experiments.

24Cellular tolerance studies. Tolerance towards NTG in LLC-PK1 cells was induced as

25previously described (Hinz and Schroder, 1998). Briefly, cells subcultured in 35-mm culture

26dishes were grown to confluence, and preincubated in basal salt solution (130 mM NaCl, 5.4 mM

27KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 5.5 mM glucose, and 20 mM HEPES-NaOH, buffered to

28pH 7.3) for 5 hours containing either NTG (1 or 100 M) or vitamin C (10 mM) or both, with

29appropriate solvent controls. At the end of the preincubation, cells were washed twice with

30phosphate-buffered saline (PBS), and further incubated in basal salt solution in the presence of

310.5 mM isobutylmethylxanthine (IBMX) for 10 min to inhibit the degradation of cGMP. NTG

32(3.16 M) was then added and incubated for another 10 min. cGMP levels in the cells were

33determined by enzyme immunoassay kit. Control experiments were conducted by preincubating

34the cells with 1 M S-nitroso-N-acetylpenicillamine (SNAP) for 5 hours followed by a

35stimulation dose of NTG (3.16 M) for cGMP measurement. Where appropriate, protein content

36was determined (Lowry et al., 1951) and used to normalize the results.

9 38To investigate the effect of GSSG on NTG induced-cGMP response, LLC-PK1 cells were pre-

39treated with 5 M - 5 mM GSSG for 1 hour. After washing twice with PBS, the cells were

40incubated with 3.16 M of NTG for 10 min. cGMP levels were determined as previously

41described.

43In the GRX transfection study, cells were preincubated in basal salt solution containing IBMX in

44the presence of vehicle or 0.01- 0.1 M NTG. After a 3-hour incubation, the cells were washed

45twice with PBS and challenged with NTG. Cells were collected for cGMP assay and protein

46determination.

48Protein 35S-cysteine incorporation determination. Cells grown to confluence in 35-mm culture

49dishes were washed twice with PBS. They were preincubated for 24 hours with 2 ml of

50Dulbecco’s Modified Eagle Medium (DMEM) without L-methionine and L-cysteine to deplete

51endogenous glutathione. After the preincubation, cells were washed twice with PBS and

52incubated with 2 ml of basal salt solution in the presence of NTG (1 or 100 M), vehicle (50%

53ethanol and 50% propylene glycol, diluted to the same extent as NTG), or 10 mM vitamin C. L-

5435S-cysteine (1 Ci/ml) was added to all samples to provide an external thiol source. After 5

55hours, the medium was discarded and the cells were washed three times with PBS. They were 10 Supplement to Role of Glutaredoxin-Mediated Protein S-Glutathionylation in Cellular Nitroglycerin Tolerance

12 56then incubated with 2 ml of DMEM containing 50 mM N-ethylmaleimide (NEM) for 5 min to

57prevent S-thiolation during the extraction process. After washing the cells 3 times with PBS, the

58cells were detached from the dish by trypsinization, and transferred to microcentrifuge tubes.

59After centrifugation (600  g for 4 min), the cells were washed 3 times with PBS containing 50

60mM NEM , and lysed with lysis buffer. Cell debris was removed by centrifugation for 20 min. A

61portion of the cell lysate supernatant was removed and mixed with 2 ml of scintillation fluid.

62This fraction was used to estimate the degree of total 35S cellular uptake. The proteins in the

63supernatant were then precipitated by adding 10% trichloroacetic acid (TCA), collected by

64centrifugation at about 10,000  g for 5 min, and the pellet was re-suspended in TCA and

65centrifuged. This procedure was repeated three times to wash away all non-protein-bound 35S.

66The protein pellet was then solubilized in 10% sodium dodecyl sulfate (SDS) solution, and 100

67l was taken out to mix with 2 ml of scintillation fluid. The mixture was then counted using a

68liquid scintillation counter (Hewlett Packard TRI-CARB 1900CA) and the extent of protein 35S-

69cysteine incorporation was estimated The rest of the sample was stored for protein determination

70and autoradiography.

72For autoradiography studies, equal amount of TCA-precipitated radio-labeled proteins were

73mixed with the gel loading buffer (1.75% SDS, 65 mM Tris-HCl, pH 6.8, 5% (v/v) glycerol, and 13 Supplement to Role of Glutaredoxin-Mediated Protein S-Glutathionylation in Cellular Nitroglycerin Tolerance

15 740.3 mM bromophenol blue) and separated by SDS-PAGE. After separation, the gel was fixed

75using 10:20:70 acetic acid: methanol: water for 1 hour. The gel was then dried for 1 hour by a gel

76drier and placed in cassette exposed to a storage phosphor screen. After 2 days of exposure,

77detection was performed on a cyclone storage phosphor system (Packard Instruments, Waltham,

78MA), and quantified using Kodak Molecular Imaging software, v.4.0.5.

80Western blot analysis. Cells preincubated with NTG (1 or 100 M) or GSSG (50 M) were

81washed two times with PBS and lysed in lysis buffer containing 2% triton X and protease

82inhibitor cocktail (1:10). Cell lysates were centrifuged for 10 min at maximum speed to remove

83cell debris and nuclei, and protein content determined. Samples containing an equal amount of

84protein was denatured in SDS non-reducing loading buffer at 90oC for 5 min, resolved in 12%

85SDS-PAGE, and electro-transferred to an ECL nitrocellulose membrane using a Mini-

86PROTEAN II Cell system (Bio-Rad, Hercules, CA). After the membrane was blocked with

87buffer (PBS containing 0.05% tween 20 and 1% bovine serum albumin) for 1 hour at room

88temperature, it was exposed to a mouse monoclonal antibody against S-glutathionylated proteins

89(1:250) overnight at 4oC. After washing, the membrane was then incubated with anti-mouse IgG

90HRP-linked conjugates (1:25,000) for 1 hour. The blot was visualized by ECL-enhanced

91chemiluminescence system. The band intensities were captured by Kodak Image Station 16 Supplement to Role of Glutaredoxin-Mediated Protein S-Glutathionylation in Cellular Nitroglycerin Tolerance

18 922000MM, and quantified using Kodak Molecular Imaging software, v.4.0.5.

94Samples obtained from GRX-over-expression experiments were lysed using lysis buffer

95described above, separated, and electroblotted. The membrane was developed by incubating with

96anti-GRX-1 primary antibody for 1 hour and anti-rabbit IgG secondary antibody for 1 hour.

97Then, ECL western blotting detection reagents were added and the membrane was detected using

98a Kodak Imaging Station 2000MM.

100Enzyme activity assays. After tolerance induction, LLC-PK1 cells were slightly trypsinized to

101detach from culture plates, and centrifuged at 600  g for 10 min at 4o C. The resulting pellet was

102lysed with lysis buffer (2% triton X and protease inhibitor cocktail), and after centrifugation, the

103supernatant was collected for enzyme assay. CK activity was measured by a standard

104spectrophotometric assay using the CK assay cocktail. A NO donor, SNAP, (100 M), was used

105to determine the effect of NO itself on CK activity. The activity of XOR was measured as

106previously described with slight modifications (Bergmeyer et al., 1974; Ichimori et al., 1999).

107The assay medium consisted of 50 mM potassium phosphate buffer (pH 7.5), 0.15 mM xanthine,

1080.2 mM EDTA, and 0.5 mM NAD+. The reaction was initiated by addition of cell lysate at room

109temperature and the appearance of NADH was monitored at 340 nm with a Shimadzu UV-1700 19 Supplement to Role of Glutaredoxin-Mediated Protein S-Glutathionylation in Cellular Nitroglycerin Tolerance

21 110spectrophotometer (Columbia, MA).

112GRX activity was measured using the low molecular weight disulfide reduction (β-

113hydroxyethylene disulfide, HED) assay developed by Holmgren and Aslund (Holmgren and

114Aslund, 1995), with a few minor modifications. Briefly, 100 mM of GSH, 20 mM NADPH, 1%

115BSA, 200 mM EDTA, 6 g/ml glutathione reductase solution, and 14 mM HED was added to 0.1

116M Tris-HCl assay buffer in a cuvette, to a final volume of 960 l. The reaction mixture was

117incubated at room temperature for two minutes to allow the mixed disulfide between HED and

118GSH to form. Then, 100 l of cell lysate was added to the cuvette and the decrease in NADPH

119absorbance was measured at 340 nm for 5 min. Total activity was calculated using the following

120equation:

121Units/mg = (∆abs/min x total volume) / (6.2 x (mg/ml of protein sample x volume of sample)).

122All samples were corrected for spontaneous background reaction, i.e. spontaneous degradation of

123NADPH. The absorption per minute of a blank control sample, i.e. when water was added in

124place of 100 l of sample, was measured and subtracted from all experimental samples. This

125value was approximately -0.020/min.

127Superoxide measurement. Flow cytometry was used to quantify the fluorescence signals

• 128produced by DHE- O2 ¯ complexes in cells. LLC-PK1 cells were incubated in blank F-12 22 Supplement to Role of Glutaredoxin-Mediated Protein S-Glutathionylation in Cellular Nitroglycerin Tolerance

24 129medium for 24 hours to stop proliferation. After suspending the cells in fresh F-12 blank medium

130(5 × 106 cells in 2 ml medium), GSSG was added to a final concentration of 50 M and

131incubated for 1 hour. Tiron and oxypurinol was added to produce final concentrations of 10mM

132and 100M, respectively. At the end of the incubation, cells were washed and suspended in 2ml

133PBS. After treating the cells with 10 nM DHE for 20 min, cells were washed with ice-cold PBS.

134They were then resuspended in 2 ml sterile PBS and analyzed using a Becton Dickinson

135FACScalibur flow cytometer with excitation and emission setting at 488 nm and 585 nm,

136respectively. BD CellQuest Pro software was used to quantify the fluorescence signal.

137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154References 155 156Bergmeyer H, Gawehn K and Grassl M (1974) Methods of enzymatic analysis. Academic Press 25 Supplement to Role of Glutaredoxin-Mediated Protein S-Glutathionylation in Cellular Nitroglycerin Tolerance

27 157 Inc., New York.

158Hinz B and Schroder H (1998) Vitamin C attenuates nitrate tolerance independently of its 159 antioxidant effect. FEBS Lett 428:97-99.

160Holmgren A and Aslund F (1995) Glutaredoxin. Methods Enzymol 252:283-292.

161Ichimori K, Fukahori M, Nakazawa H, Okamoto K and Nishino T (1999) Inhibition of xanthine 162 oxidase and xanthine dehydrogenase by nitric oxide. Nitric oxide converts reduced 163 xanthine-oxidizing enzymes into the desulfo-type inactive form. J Biol Chem 274:7763- 164 7768.

165Lowry OH, Rosebrough NJ, Farr AL and Randall RJ (1951) Protein measurement with the Folin 166 phenol reagent. J Biol Chem 193:265-275. 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182Supplemental Fig. 1. (A) Autoradiography of Western blots of cellular proteins after control

183(lanes C1-C3) vs. NTG exposure (lanes N1-N3). (B) Densitometric analysis of bands shown in 28 Supplement to Role of Glutaredoxin-Mediated Protein S-Glutathionylation in Cellular Nitroglycerin Tolerance

30 184panel A. Values are expressed as mean  SD, n = 3 for each group. Under tolerance condition,

18535S-incorporation was significantly more extensive in multiple proteins after NTG treatment

186(unfilled bars) vs. control (filled bars, *p<0.05, Student’s t-test), suggesting NTG-induced 35S-

187incorporation was a non-specific reaction. (C) Representative Western blot of protein S-

188glutathionylation in control and NTG treated LLC-PK1 cells. (D) Analysis of band densities of

189proteins shown in panel A. Vehicle control (filled bars), 100 M NTG (unfilled bars), 10 mM

190vitamin C (striped bars), and NTG + vitamin C (cross-hatched bars). Values are expressed as

191mean  SD, n = 4-8 for each group. Several proteins (with approximate molecular weights of 20,

19225, 33, and 40 kDa, one-way ANOVA, p<0.01; post-hoc test: *p<0.05 vs. control,) were S-

193glutathionylated when tolerance was induced, while co-incubation of vitamin C attenuated the

194extent of S-glutathionylation of several proteins (about 25, 33, and 105 kDa, one-way ANOVA,

195p<0.01; post-hoc test: #p<0.01 vs. NTG).

200 A. 201

33 203 204 205 206 207 208 209 210 211 212 213 B.

214 Protein S- Incorporation in LLC-PK1 cells 215 35000 216 30000 y

217 t i * s 25000 * n e

218 t n

I 20000

219 n a 15000 B

N 10000 221 * * * 222 5000 *

0 223 105k 75k 50k 35k 33k 30k 25k 23k 20k 17k 15k 13k 11k 224 MW on gel 225 226 227 228 229 230 231 232 233 C. 234 235 236 237 238 34 Supplement to Role of Glutaredoxin-Mediated Protein S-Glutathionylation in Cellular Nitroglycerin Tolerance

36 239 240 241 242 243 244 245 246 247 D. 248 Nitroglycerin Induced Protein S-Glutathionylation 249

250 1.6e+5 251 1.4e+5 y t i

252 s 1.2e+5 n e t 1.0e+5 *

253 I * t

e 8.0e+4 254 N * d

n 6.0e+4

255 a B 4.0e+4 256 # # 257 2.0e+4 # 258 0.0 105k 50k 40k 33k 30k 25k 20k 259 MW on gel Each group analyzed by One-Way Anova with Post hoc *p<0.05 vs. Control 260 #p<0.01 vs. Nitroglycerin n=8 for Control and Nitroglycerin 261 n=4 for vitamin C and Nitroglycerin + Vitamin C 262 263 264 265

267Supplemental Fig. 2. Representative Western blot (A) and densitometry analysis (B) of protein

268S-glutathionylation in LLC-PK1 cells treated with control (unfilled bars), 50 M GSSG (unfilled

269bars), 10 mM vitamin C (striped bars), and GSSG + vitamin C (crossed hatched bars). Values are 37 Supplement to Role of Glutaredoxin-Mediated Protein S-Glutathionylation in Cellular Nitroglycerin Tolerance

39 270expressed as mean  SD, n = 4 for each group. Several proteins (with molecular weights of 20,

27123, 33, and 40 kDa, one-way ANOVA, p<0.05; post-hoc test: *p<0.05 vs. control) were S-

272glutathionylated when treated with GSSG, while co-incubation of vitamin C attenuated the

273extent of S-glutathionylation (proteins of 25, 33, and 40 kDa, one-way ANOVA, p<0.05; post-

274hoc test: #p<0.01 vs. GSSG).

275 A. 276 277 278 279 280 281 282 283 284 285 286 B. Oxidized Glutathione Induced Protein S-Glutathionylation 287 1.6e+5

288 1.4e+5 y

t 1.2e+5 289 i * s n

e 1.0e+5 t n I

e 8.0e+4 * N

291 d *

n 6.0e+4 a

B # # 4.0e+4 # 2.0e+4

0.0 105k 50k 40k 33k 25k 23k 20k MW on gel

Each group analyzed by One-Way Anova with Post hoc *p<0.05 vs. Control #p<0.01 vs. GSSG n=4 for each group