Detection and Imaging of Nitric Oxide with Novel Fluorescent Indicators: Diaminofluoresceins

Article abstract of DOI:10.1021/ac9801723

Nitric oxide is a gaseous, free radical which plays a role as an intracellular second messenger and a diffusable intercellular messenger. To obtain direct evidence for NO functions in vivo, we have designed and synthesized diaminofluoresceins (DAFs) as no

Full text of DOI:10.1021/ac9801723

Anal. Chem. 1998, 70, 2446-2453
Detection and Imaging of Nitric Oxide with Novel
Fluorescent Indicators: Diaminofluoresceins
Hirotatsu Kojima,† Naoki Nakatsubo,† Kazuya Kikuchi,† Shigenori Kawahara,† Yutaka Kirino,†
Hiroshi Nagoshi,‡ Yasunobu Hirata,‡ and Tetsuo Nagano*,†
Graduate School of Pharmaceutical Sciences and Second Department of Internal Medicine, Graduate School of Medicine,
the University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
of selectivity, and experimental feasibility. Experimental studies
on Ca2+-dependent signal transduction in cells were greatly
facilitated by the development of fluorescent indicators for
Nitric oxide is a gaseous, free radical which plays a role
as an intracellular second messenger and a diffusable
intercellular messenger. To obtain direct evidence for NO
functions in vivo, we have designed and synthesized
diaminofluoresceins (DAFs) as novel fluorescent indica-
tors for NO. The fluorescent chemical transformation of
DAFs is based on the reactivity of the aromatic vicinal
diamines with NO in the presence of dioxygen. The
N-nitrosation of DAFs, yielding the highly green-fluores-
cent triazole form, offers the advantages of specificity,
sensitivity, and a simple protocol for the direct detection
of NO (detection limit 5 nM). The fluorescence quantum
efficiencies are increased more than 1 0 0 times after the
transformation of DAFs by NO. Fluorescence detection
with visible light excitation and high sensitivity enabled
the practical assay of NO production in living cells.
Membrane-permeable DAF-2 diacetate (DAF-2 DA) can
be used for real-time bioimaging of NO with fine temporal
and spatial resolution. The dye was loaded into activated
rat aortic smooth muscle cells, where the ester bonds are
hydrolyzed by intracellular esterase, generating DAF-2 .
The fluorescence in the cells increased in a NO concen-
tration-dependent manner.
cytosolic Ca2+ 8,9
Although bioimaging of NO has already been
.
reported by chemiluminescence10 and ESR,11 they have disadvan-
tages to the functional analysis of NO, such as the use of cytotoxic
H2O2 or low spatial resolution. The use of NO-reactive fluorescent
indicators, in conjunction with confocal laser microscopy, should
make possible the bioimaging of NO, which is suitable for real-
time analysis of intracellular NO. The fluorescence detection of
intracellular NO based on the conversion of 2,7-dichlorofluorescin
was reported; however, the system is unable to distinguish
between NO and reactive oxygen species, and the limit of the
detection of NO is 16 µM.12 We therefore newly designed and
synthesized fluorescent NO indicators to detect NO in living cells
as a means to examine the physiological functions of NO. First,
the reactivity of NO was examined in order to find a suitable
reaction for selective NO trapping. Wink et al. reported that NO
deaminates deoxynucleosides, deoxynucleotide, and intact DNA
at physiological pH.13 We found that aromatic amines react with
NO in the presence of dioxygen to produce the corresponding
triazenes,14 and the corresponding triazole ring compounds are
generated from aromatic vicinal diamines under neutral conditions.
The reaction mechanism is proposed to involve nitrous anhydride,
which is generated according to the following scheme:15
Nitric oxide is a messenger molecule involved in the regulation
of diverse physiological and pathophysiological mechanisms in
the cardiovascular system, the central and peripheral nervous
systems, and the immune system.1-3 However, many proposed
physiological roles of NO were not proved directly by measuring
NO. One of the reasons for this is the difficulty of direct, real-
time detection of this gaseous, free-radical species. Although
several methods of detecting NO, which is unstable and produced
at low concentration, have been developed,4-7 a new method is
required which is satisfactory for studies in living cells in terms
2NO + O2 f 2NO2
(1)
(7) Kikuchi, K.; Nagano, T.; Hayakawa, H.; Hirata, Y.; Hirobe, M. J. Biol. Chem.
1 9 9 3 , 268, 23106-23110.
(8) Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. J. Biol. Chem. 1 9 8 5 , 260, 3440-
3450.
(9) Minta, A.; Kao, J. P. Y.; Tsien, R. Y. J. Biol. Chem. 1 9 8 9 , 264, 8171-8178.
(10) Leone, A. M.; Furst, V. W.; Foxwell, N. A.; Cellek, S.; Moncada, S. Biochem.
Biophys. Res. Commun. 1 9 9 6 , 221, 37-41.
* Corresponding author: (e-mail) tlong@mol.f.u-tokyo.ac.jp; (fax) 81-3-5684-
2395.
(11) Yoshimura, T.; Yokoyama, H.; Fujii, S.; Takayama, F.; Oikawa, K.; Kamada,
H. Nature Biotechnol. 1 9 9 6 , 14, 992-994.
(12) Gunasekar, P. G.; Kanthasamy, A. G.; Borowitz, J. L.; Isom, G. E. J. Neurosci.
Methods 1 9 9 5 , 61, 15-21.
† Graduate School of Pharmaceutical Sciences.
‡ Second Department of Internal Medicine.
(1) Moncada, S.; Higgs, A. N. Engl. J. Med. 1 9 9 3 , 329, 2002-2012.
(2) Snyder, S. H.; Bredt, D. S. Sci. Am. 1 9 9 2 , 266, 68-77.
(3) Nathan, C.; Xie, Q. Cell 1 9 9 4 , 78, 915-918.
(4) Malinski, T.; Mesaros, S.; Tomboulian, P. Methods Enzymol. 1 9 9 6 , 268,
58-69.
(13) Wink, D. A.; Kasprzak, K. S.; Maragos, C. M.; Elespuru, R. K.; Misra, M.;
Dunams, T. M.; Cebula, T. A.; Koch, W. H.; Andrews, A. W.; Allen, J. S.;
Keefer, L. K. Science 1 9 9 1 , 254, 1001-1003.
(14) Nagano, T.; Takizawa, H.; Hirobe, M. Tetrahedron Lett. 1 9 9 5 , 36, 8239-
8242.
(5) Palmer, R. M. J.; Ferrige, A. G.; Moncada, S. Nature 1 9 8 7 , 327, 524-526.
(6) Akaike, T.; Maeda, H. Methods Enzymol. 1 9 9 6 , 268, 211-221.
(15) Ignarro, L. J.; Fukuto, J. M.; Griscavage, J. M.; Rogers, N. E.; Byrns, R. E.
Proc. Natl. Acad. Sci. U.S.A. 1 9 9 3 , 90, 8103-8107.
2446 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
S0003-2700(98)00172-3 CCC: $15.00 © 1998 American Chemical Society
Published on Web 05/28/1998

(s, 3H, COCH3), 2.39 (s, 3H, CH3), 7.18 (d, 1H, ArH, J ) 8.3 Hz),
7.80 (d, 1H, ArH, J ) 8.3 Hz), 8.46 (s, 1H, NH).
2NO + 2NO T 2N2O3
(2)
II. A suspension of potassium permanganate (133 g, 841
mmol) in water (1 L) was added in several portions, as dissolution
of the solid proceeded, to a suspension of I (29.2 g, 140 mmol) in
hot water (1 L) with magnesium sulfate (100 g, 831 mmol). The
resulting brown suspension was filtered hot, and the filtrate was
saturated with NaCl. After cooling to room temperature, the
yellow filtrate was acidified with HCl and the cream-colored
precipitate was collected. In addition, the filtrate was extracted
with ethyl acetate and the extract was worked up in the usual
way (19.6 g, 52% yield): 1H NMR (dimethyl sulfoxide (DMSO)-
d6) δ 1.97 (s, 3H, COCH3), 7.90 (d, 1H, ArH, J ) 8.4 Hz), 8.04 (d,
1H, ArH, J ) 8.4 Hz), 10.13 (s, 1H, NH), 13.5 (br, 2H, COOH).
III. Acetyl chloride (30 mL) was added to II (19.0 g, 70.8
mmol) in acetic anhydride (180 mL) at 80 °C, and the mixture
was refluxed for 2 h. The solvent was removed in vacuo, a small
amount of anhydrous dichloromethane was added, and the cream-
colored precipitate was collected (9.87 g, 56%): 1H NMR (CDCl3)
δ 2.35 (s, 3H, COCH3), 7.89 (d, 1H, ArH, J ) 8.2 Hz), 8.45 (d, 1H,
ArH, J ) 8.2 Hz), 9.01 (s, 1H, NH).
III f IV, V, VI, and VII. A mixture of 3-acetamido-4-
nitrophthalic anhydride (III) and resorcinol (or 4-chlororesorcinol)
(2 equiv) was stirred at 180 °C for 2 h. After the addition of ZnCl2
(0.25 equiv), the temperature was raised to 210 °C and stirring
was continued until a dry residue was obtained. After cooling,
the black powder was refluxed in 0.6 M HCl (containing 1 equiv
of HCl) for 1 h.21 The black solid was collected by filtration and
dried. The products were purified by silica gel chromatography
eluted with methanol/ dichloromethane (IV, V, the concentration
of methanol was gradually raised from 7 to 15% v/ v; IV, V, the
concentration of methanol was gradually raised from 3 to 10% v/ v),
to afford IV (5%) and V (2%) (or VI (6%) and VII (1%)): 1H NMR
(DMSO-d6) (IV) δ 6.38 (d, 1H, ArH, J ) 8.6 Hz), 6.56 (dd, 2H,
ArH, J ) 8.6, 2.4 Hz), 6.66 (d, 2H, ArH, J ) 2.4 Hz), 6.80 (d, 2H,
ArH, J ) 8.6 Hz), 7.96 (s, 2H, NH2), 8.35 (d, 1H, ArH, J ) 8.6 Hz),
10.18 (s, 2H, OH); (V) δ 6.40 (s, 2H, NH2), 6.56 (dd, 2H, ArH, J
) 8.6, 2.4 Hz), 6.71 (d, 2H, ArH, J ) 2.4 Hz), 6.72 (d, 2H, ArH, J
) 8.6 Hz), 7.22 (d, 1H, ArH, J ) 8.6 Hz), 8.38 (d, 1H, ArH, J ) 8.6
Hz), 10.18 (s, 2H, OH); (VI) δ 6.41 (d, 1H, ArH, J ) 8.6 Hz), 6.87
(s, 2H, ArH), 7.03 (s, 2H, ArH), 7.83 (s, 2H, NH2), 8.36 (d, 1H,
ArH, J ) 8.6 Hz), 11.23 (s, 2H, OH). VII: δ 6.60 (s, 2H, NH2),
6.93 (s, 2H, ArH), 6.93 (s, 2H, ArH), 7.22 (d, 1H, ArH, J ) 8.6
Hz), 8.40 (d, 1H, ArH, J ) 8.6 Hz), 11.17 (s, 2H, OH).
We started to investigate the design and synthesis of fluorescent
compounds based originally on 2,3-diaminonaphthalene as a vicinal
diamine. During this work, a similar method was reported.16 We
developed a new fluorescent indicator (DAN-1 EE) for bioimaging
of NO17 and found some problems, such as cytotoxcity and strong
autofluorescence owing to the requirement for excitation for UV
light, small extinction coefficient, and poor solubility in neutral
buffer.
Fluorescein is widely used in biology as a fluorophore because
of its convenient wavelengths for biological measurement, high
extinction coefficient, and high fluorescence quantum yield in
water. Fluoresceinamine was reported to show quenched fluo-
rescence, but the conversion of amine to amide resulted in
fluorescent properties.18 This result implies that the fluorescence
was quenched because of the electron-donating group attached
to the phthalic ring of fluorescein. However, when the electron-
donating group was transformed into a less electron-donating
group, the fluorescence recovered. Based on these findings, we
designed diaminofluoresceins (DAFs, Figure 1) as indicators for
NO. In other words, we considered that the electron donation of
the functional groups attached to fluorescein would be reduced
by reaction with NO via triazole ring formation, leading to an NO
concentration-dependent enhancement of fluorescence.
In order to load the cells efficiently with the probe, it was
necessary to prepare the diacetate (DAF-2 DA, Figure 1). It can
permeate readily into the cells, where it is hydrolyzed by
intracellular esterases existing in many cell types, to generate
DAF-2.19 Escherichia coli lipopolysaccharide (LPS) and certain
kinds of cytokines cause an increase in an inducible type of NO
synthase in vascular smooth muscle cells.20 We evaluated this
new probe for real-time biological imaging of NO in cultured rat
aortic smooth muscle cells.
EXPERIMENTAL SECTION
Synthesis of DAFs. 1H NMR spectra were recorded on a
JEOL JNM-LA300 instrument at 300 MHz; δ values in ppm relative
to tetramethylsilane are given. Mass spectrometry (MS) spectra
were measured with JEOL JMS-DX 300 mass spectrometer. IR
spectra were measured on a Jasco FT-IR-5300 instrument. The
synthetic pathways employed to obtain the new dyes are outlined
in Figure 1.
IV f DAF-1, V f DAF-3, VI f DAF-4, and VII f DAF-6.
Aminonitrofluoresceins were reduced with Na2S and NaSH in
water, as described by McKinney et al.,22 to give diaminofluores-
ceins (DAF-1, 76%; DAF-3, 30%; DAF-4, 52%; DAF-6, 16%). DAFs
were purified by silica gel chromatography eluted with methanol/
dichloromethane (DAF-1, 1:9; DAF-3, 7:93; DAF-4, 5:95; DAF-6,
5:95 v/ v) and recrystallized from isopropyl alcohol. (DAF-1) mp
I. Acetic anhydride (15 mL, 0.16 mol) was added to a solution
of 2,3-dimethyl-6-nitroaniline (25.3 g, 0.152 mol) in hot glacial
acetic acid (290 mL). The mixture was refluxed for 1 h, acetic
acid was removed by evaporation, and the residue was recrystal-
lized from ethanol to give a quantitative yield of 2,3-dimethyl-6-
nitroacetanilide (I): 1H NMR (CDCl3) δ 2.18 (s, 3H, CH3), 2.24
1
(16) Miles, A. M.; Wink, D. A.; Cook, J. C.; Grisham, M. B. Methods Enzymol.
1 9 9 6 , 268, 105-120.
(17) Kojima, H.; Sakurai, K.; Kikuchi, K.; Kawahara, S.; Kirino, Y.; Nagoshi, H.;
Hirata, Y.; Akaike, T.; Maeda, H.; Nagano, T. Biol. Pharm. Bull. 1 9 9 7 , 20,
1229-1232.
>300 °C; H NMR (DMSO-d6) δ 5.01 (s, 2H, NH2), 5.88 (s, 2H,
NH2), 6.05 (d, 1H, ArH, J ) 7.5 Hz), 6.52 (dd, 2H, ArH, J ) 8.6,
2.4 Hz), 6.60 (d, 2H, ArH, J ) 2.4 Hz), 6.64 (d, 2H, ArH, J ) 8.6
Hz), 6.78 (d, 1H, ArH, J ) 7.5 Hz), 9.97 (s, 2H, OH); MS (EI),
(18) Munkholm, C.; Parkinson, D.-R.; Walt, D. R. J. Am. Chem. Soc. 1 9 9 0 , 112,
2608-2612.
(21) Kametani, T. The Chemistry of Organic Synthesis; Nankodo: Tokyo, 1979;
(19) Rotman, B.; Papermaster, B. W. Proc. Natl. Acad. Sci. U.S.A. 1 9 6 6 , 55,
134-141.
(20) Busse, R.; Mu¨ lsch, A. FEBS Lett. 1 9 9 0 , 275, 87-90.
Vol. IX, p 215.
(22) McKinney, R. M.; Spillane, J. T.; Pearce, G. W. J. Org. Chem. 1 9 6 2 , 27,
3986-3988.
Analytical Chemistry, Vol. 70, No. 13, July 1, 1998 2447

Figure 1. Synthesis of DAFs. Roman numerals correspond to the synthetic details given under Experimental Section. XI and XII represent
mixtures of isomers.
m/ z (M+) 362; IR (KBr) 1730 (CdO), 1615 (aromatic), 1453
(aromatic) cm-1
Elemental Anal., Calcd for C20H14N2O5‚
NH2), 6.12 (d, 1H, ArH, J ) 7.7 Hz), 6.68 (s, 2H, ArH), 6.81 (d,
1H, ArH, J ) 7.7 Hz), 6.85 (s, 2H, ArH), 10.97 (s, 2H, OH); MS
(EI), m/ z (M+) 430; IR (KBr) 1705 (CdO), 1610 (aromatic), 1381
.
2C3H8O: C, 64.71; H, 6.27; N, 5.81. Found: C, 64.62; H, 6.21; N,
1
5.48. (DAF-3) mp 220-230 °C; H NMR (DMSO-d6) δ 3.80 (s,
(aromatic) cm-1
.
Elemental Anal., Calcd for C20H12-
2H, NH2), 5.58 (s, 2H, NH2), 6.53 (dd, 2H, ArH, J ) 8.6, 2.4 Hz),
6.62 (d, 2H, ArH, J ) 8.6 Hz), 6.65 (d, 2H, ArH, J ) 2.4 Hz), 6.79
(d, 1H, ArH, J ) 7.9 Hz), 7.07 (d, 1H, ArH, J ) 7.9 Hz), 10.01 (s,
2H, OH); MS (EI), m/ z (M+) 362; IR (KBr) 1730 (CdO), 1611
Cl2N2O5‚C3H8O: C, 56.22; H, 4.10; N, 5.70. Found: C, 55.82; H,
4.15; N, 5.79. (DAF-6) mp >300 °C (sublimed at 230 °C); 1H NMR
(DMSO-d6) δ 4.06 (s, 2H, NH2), 5.74 (s, 2H, NH2), 6.61 (s, 2H,
ArH), 6.84 (d, 1H, ArH, J ) 7.9 Hz), 6.89 (s, 2H, ArH), 7.10 (d,
1H, ArH, J ) 7.9 Hz), 10.97 (s, 2H, OH); MS (EI), m/ z (M+) 430;
(aromatic), 1453 (aromatic), 1383 (aromatic) cm-1
. Elemental
Anal., Calcd for C20H14N2O5‚1.4H2O: C, 61.98; H, 4.37; N, 7.23.
Found: C, 61.70; H, 4.09; N, 7.18. (DAF-4) mp >300 °C (sublimed
at 240 °C); 1H NMR (DMSO-d6) δ 5.14 (s, 2H, NH2), 5.97 (s, 2H,
IR (KBr) 1719 (CdO), 1456 (aromatic), 1385 (aromatic) cm-1
.
Elemental Anal., Calcd for C20H12Cl2N2O5‚C3H8O: C, 56.22; H, 4.10;
N, 5.70. Found: C, 55.97; H, 4.02; N, 6.00.
2448 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

VIII. Acetic anhydride (10 mL, 0.11 mol) was added to a
solution of 4,5-dimethyl-2-nitroaniline (17.1 g, 103 mmol, Aldrich)
in hot acetic acid (200 mL). The mixture was refluxed for 1 h,
acetic acid was removed by evaporation, and the residue was
recrystallized from ethanol to afford a quantitative yield of
4-acetamido-5-nitroxylene (VIII): 1H NMR (CDCl3) δ 2.27 (s, 3H,
COCH3), 2.28 (s, 3H, CH3), 2.35 (s, 3H, CH3), 7.98 (s, 1H, ArH),
8.54 (s, 1H, ArH), 10.30 (s, 1H, NH).
IX. A suspension of potassium permanganate (42.4 g, 268
mmol) in water (300 mL) was added dropwise to a suspension of
VIII (11.8 g, 56.7 mmol) in hot water (700 mL) with magnesium
sulfate (42.4 g, 352 mmol). The resulting brown suspension was
filtered hot, and the yellow filtrate was acidified with HCl and
extracted with ethyl acetate. 4-Acetamido-5-nitrophthalic acid (IX)
was obtained after evaporation (8.9 g, 59%): 1H NMR (DMSO-d6)
δ 2.10 (s, 3H, COCH3), 7.91 (s, 1H, ArH), 8.26 (s, 1H, ArH), 10.53
(s, 1H, NH), 13.0 (br, 2H, COOH).
gel chromatography eluted with methanol/ dichloromethane (3:
97 v/ v) to afford 4,5-diaminofluorescein diacetate (DAF-2 DA) (137
mg, 90%): mp 110-120 °C; H NMR (acetone-d6) δ 2.27 (s, 6H,
1
COCH3), 4.68 (s, 2H, NH2), 5.16 (s, 2H, NH2), 6.34 (s, 1H, ArH),
6.92 (dd, 2H, ArH, J ) 8.6, 2.2 Hz), 7.00 (d, 2H, ArH, J ) 8.6 Hz),
7.12 (d, 2H, ArH, J ) 2.2 Hz), 7.15, (s, 1H, ArH); MS (EI), m/ z
446 (M+). IR (KBr) 1750 (lactone), 1606 (aromatic) cm-1
.
Elemental Anal., Calcd for C24H18N2O7: C, 64.57; H, 4.06; N, 6.28.
Found: C, 64.27; H, 4.16; N, 6.18.
Triazole Forms of DAFs. NO gas was introduced into a
methanol solution of each DAF to yield the corresponding
triazolofluorescein (DAF-1 T-DAF-6 T). Each triazolofluorescein
was purified by silica gel chromatography eluted with methanol/
dichloromethane (1:9 v/ v, a small amount of acetic acid (0.02%
v/ v) was added only in the case of purification of DAF-4 T), and
the dried residue was dissolved in 2 M NaOH. Then the solution
was adjusted to pH 3-4 with HCl, and the precipitate was collected
and dried. (DAF-1 T) mp >300 °C; 1H NMR (acetone-d6) δ 6.60
(dd, 2H, ArH, J ) 8.6, 2.4 Hz), 6.77 (d, 2H, ArH, J ) 2.4 Hz), 6.77
(d, 2H, ArH, J ) 8.6 Hz), 7.23 (d, 1H, ArH, J ) 7.7 Hz), 8.43 (d,
1H, ArH, J ) 7.7 Hz), 9.05 (s, 2H, OH); MS (EI), m/ z (M+) 373;
X. IX (6.0 g, 22 mmol) was dehydrated in acetyl chloride (60
mL) under reflux.23 Acetyl chloride was removed, and the
recrystallization of the residue from benzene gave 4-acetamido-
5-nitrophthalic anhydride (X) (5.0 g, 90%): 1H NMR (CDCl3) δ
2.40 (s, 3H, COCH3), 8.85 (s, 1H, ArH), 9.54 (s, 1H, ArH), 10.64
(s, 1H, NH).
IR (KBr) 1734 (CdO), 1615 (aromatic), 1453 (aromatic) cm-1
.
1
(DAF-2 T) mp >300 °C; H NMR (acetone-d6) δ 6.59 (dd, 2H,
ArH, J ) 8.8, 2.4 Hz), 6.72 (d, 2H, ArH, J ) 8.8 Hz), 6.75 (d, 2H,
ArH, J ) 2.4 Hz), 7.68 (s, 1H, ArH), 8.56 (s, 1H, ArH); MS (EI),
m/ z (M+) 373; IR (KBr) 1725 (CdO), 1616 (aromatic), 1474
X f XI and XII. XI and XII were synthesized from X by the
same method as used to obtain IV-VII (XI, A 34%, B 19%. XII,
A + B 30% mixture after solidification was used for the next
reaction): 1H NMR (DMSO-d6) (XI-A) δ 6.58 (dd, 2H, ArH, J )
8.6, 2.2 Hz), 6.63 (s, 1H, ArH), 6.65 (d, 2H, ArH, J ) 2.2 Hz), 6.75
(d, 2H, ArH, J ) 8.6 Hz), 7.92 (s, 2H, NH2), 8.49 (s, 1H, ArH),
10.18 (s, 2H, OH). (XI-B) δ 6.55 (dd, 2H, ArH, J ) 8.6, 2.2 Hz),
6.65 (d, 2H, ArH, J ) 2.2 Hz), 6.74 (d, 2H, ArH, J ) 8.6 Hz), 7.59
(s, 1H, ArH), 7.73 (s, 2H, NH2), 7.74 (s, 1H, ArH), 10.12 (s, 2H,
OH). (XII-A) δ 6.66 (s, 1H, ArH), 6.89 (s, 2H, ArH), 6.91 (s, 2H,
ArH), 7.96 (s, 2H, NH2), 8.48 (s, 1H, ArH), 11.11 (s, 2H, OH).
XII-B: δ 6.9 (br, 2H, ArH), 6.91 (s, 2H, ArH), 7.63 (s, 1H, ArH),
7.77 (s, 2H, NH2), 7.86 (s, 1H, ArH), 11.07 (s, 2H, OH).
XI f DAF-2 and XII f DAF-5. These compounds were
synthesized from XI or XII by a method similar to that used for
DAF-1, 3, 4, and 6 (DAF-2, from XI-A 97%, XI-B 30%. DAF-5, from
1
(aromatic), 1383 (aromatic) cm-1. (DAF-3 T) mp >300 °C; H
NMR (acetone-d6) δ 6.54 (dd, 2H, ArH, J ) 8.6, 2.4 Hz), 6.67 (d,
2H, ArH, J ) 8.6 Hz), 6.79 (d, 2H, ArH, J ) 2.4 Hz), 8.00 (d, 1H,
ArH, J ) 8.0 Hz), 8.24 (d, 1H, ArH, J ) 8.0 Hz), 9.00 (s, 2H, OH);
MS (EI), m/ z (M+) 373; IR (KBr) 1734 (CdO), 1611 (aromatic),
1474 (aromatic), 1383 (aromatic) cm-1. (DAF-4 T) mp >300 °C;
1H NMR (acetone-d6) δ 6.97 (s, 2H, ArH), 7.00 (s, 2H, ArH), 7.31
(d, 1H, ArH, J ) 9.0 Hz), 8.46 (d, 1H, ArH, J ) 9.0 Hz), 9.70 (s,
2H, OH); MS (EI), m/ z (M+) 441; IR (KBr) 1748 (CdO), 1582
(aromatic), 1491 (aromatic), 1385 (aromatic) cm-1. (DAF-5 T)
1
mp >300 °C; H NMR (acetone-d6) δ 6.86 (s, 2H, ArH), 6.93 (s,
2H, ArH), 7.8 (br, 1H, ArH), 8.6 (br, 1H, ArH), 9.62 (s, 2H, OH);
MS (EI), m/ z (M+) 441; IR (KBr) 1724 (CdO), 1385 (aromatic)
1
1
XII-A + B (mixture) 53%). (DAF-2) mp 240-250 °C; H NMR
cm-1. (DAF-6 T) mp >300 °C; H NMR (acetone-d6) δ 6.88 (s,
(DMSO-d6) δ 5.00 (s, 2H, NH2), 5.58 (s, 2H, NH2), 6.07 (s, 1H,
ArH), 6.52 (dd, 2H, ArH, J ) 8.6, 2.2 Hz), 6.60 (d, 2H, ArH, J )
2.2 Hz), 6.60 (d, 2H, ArH, J ) 8.6 Hz), 6.89 (s, 1H, ArH), 9.99 (s,
2H, OH); MS (EI), m/ z (M+) 362; IR (KBr) 1732 (CdO), 1618
2H, ArH), 7.00 (s, 2H, ArH), 8.04 (d, 1H, ArH, J ) 8.0 Hz), 8.23
(d, 1H, ArH, J ) 8.0 Hz), 9.68 (s, 2H, OH); MS (EI), m/ z (M+)
441; IR (KBr) 1717 (CdO), 1636 (aromatic), 1458 (aromatic), 1385
(aromatic) cm-1
.
(aromatic), 1458 (aromatic), 1383 (aromatic) cm-1
.
Elemental
P reparation of NO Donor and NO Solution. The
Anal., Calcd for C20H14N2O5‚2C3H8O: C, 64.71; H, 6.27; N, 5.81.
Found: C, 64.67; H, 6.15; N, 6.08. (DAF-5) mp 215-220 °C; H
NONOates were prepared according to the methods of Hrabie
1
and his associates.24 The structures of NOC12 and NOC13 are
+
NMR (DMSO-d6) δ 5.12 (s, 2H, NH2), 5.69 (s, 2H, NH2), 6.12 (s,
1H, ArH), 6.63 (s, 2H, ArH), 6.85 (s, 2H, ArH), 6.93 (s, 1H, ArH),
10.96 (s, 2H, OH); MS (EI), m/ z (M+) 430; IR (KBr) 1717 (CdO)
cm-1. Elemental Anal., Calcd for C20H12Cl2N2O5‚C3H8O: C, 56.22;
H, 4.10; N, 5.70. Found: C, 56.22; H, 3.98; N, 5.84.
DAF-2 f DAF-2 DA. Cesium carbonate (166 mg, 0.509 mmol)
was added to an acetonitrile solution (100 mL) of DAF-2 (124 mg,
0.341 mmol) and then acetic anhydride (105 µL, 1.12 mmol) was
added dropwise. The mixture was stirred for 1 h, the solvent was
removed by evaporation, and the residue was subjected to silica
EtN[N(O)NO]-(CH2)2NH2+Et and MeN[N(O)NO]-(CH2)3NH3
respectively. NO solution was prepared by bubbling NO through
sodium phosphate buffer for about 30 s. The buffer was deoxi-
dized by bubbling argon through it for 1 h before NO introduction,
and the concentration was determined by horseradish peroxidase
assay.25
,
Fluorometric Analysis. A fluorescence spectrophotometer
(F4500, Hitachi, Tokyo, Japan) was used. The slit width was 2.5
(24) Hrabie, J. A.; Klose, J. R.; Wink, D. A.; Keefer, L. K. J. Org. Chem. 1 9 9 3 ,
58, 1472-1476.
(25) Kikuchi, K.; Nagano, T.; Hirobe, M. Biol. Pharm. Bull. 1 9 9 6 , 19, 649-
(23) Goldstein, H.; Merminod, J.-P. Helv. Chim. Acta 1 9 5 2 , 35, 1476-1480.
651.
Analytical Chemistry, Vol. 70, No. 13, July 1, 1998 2449

nm for both excitation and emission. The photomultiplier voltage
was 950 V. DAFs were dissolved in DMSO to obtain 10 mM stock
solutions. Relative quantum efficiencies of fluorescence of DAFs
and triazolofluoresceins were obtained by comparing the area
under the corrected emission spectrum of the test sample at 492-
nm excitation with that of a solution of fluorescein in 0.1 M NaOH,
which has a quantum efficiency of 0.85 according to the litera-
ture.26
HP LC Analysis. The HPLC unit consisted of a pump (PU980,
Jasco), a detector (UV-970 or FU-920, Jasco), and a column
(Finepak SIL C18-5, 6.0 × 250 mm, Jasco). The eluent for analysis
was pH 7.4 sodium phosphate buffer/ 10 mM acetonitrile (94:6
v/ v). The flow rate was 1.0 mL/ min. Absorbance was monitored
at 495 nm for colorimetric assay. Fluorescence was monitored
at ex/ em 495 nm/ 515 nm. Aliquots of 10 µL were injected for
analysis. Macrophages (RAW 264.7) were cultured on 24-well
plates, and DAF was added to the culture medium. The mac-
rophages were stimulated 16 h before measurement with LPS (10
ng/ mL) and recombinant murine interferon-γ (IFN-γ) (10 units/
mL), when necessary. After 2 h, an aliquot (10 µL) of each cell
supernatant was taken and analyzed by HPLC.
Table 1. Absorbance and Fluorescence Properties of
Dyesa
extinctn coeff and
fluorescence
abs max,b × 104 M-1 cm-1
rel quantum effic
diamine triazole
max of triazole
forms, nm
DAF
diamine
triazole
1
2
3
4
5
6
8.1 (489)
7.9 (486)
8.1 (493)
9.7 (501)
9.8 (499)
11 (505)
5.6 (493)
7.3 (491)
6.6 (493)
9.2 (505)
7.9 (503)
4.3 (506)
521
513
521
530
523
528
0.004
0.005
0.005
0.002
0.007
0.003
0.78
0.92
0.85
0.75
0.70
0.53
a
All data were obtained at 20 °C in 0.1 M sodium phosphate buffer,
pH 7.4. Relative quantum efficiencies were measured as described
under Experimental Section. b Values in parentheses are nm.
P reparation of Activated Rat Aortic Smooth Muscle Cells.
Fetal bovine serum (FBS) was purchased from Bioserum (Victoria,
Australia). Recombinant murine interleukin-1â (IL-1â) and re-
combinant murine tumor necrosis factor-R (TNF-R) were pur-
chased from R&D Systems (Minneapolis, MN). IFN-γ was
purchased from Genzyme (Cambridge, MA). LPS was purchased
from Difco Laboratories (Detroit, MI).
L-Arginine (L-Arg), and
N-monomethyl- -arginine ( -NMMA) were purchased from Wako
L
L
Pure Chemical Industries (Osaka, Japan). Krebs-Ringer phos-
phate buffer (KRP) consists of 120 mM NaCl, 4.8 mM KCl, 0.54
mM CaCl2, 1.2 mM MgSO4, 11 mM glucose, and 15.9 mM sodium
phosphate (pH 7.2).
Vascular smooth muscle cells were isolated from thoracic aorta
of male Wistar rats by a standard explant method25 and grown in
Dulbecco’s modified Eagle’s medium (DME) supplemented with
10% FBS and antibiotics. Cells were incubated at 37 °C in a humid
atmosphere of 95% air/ 5% CO2 on glass-bottomed culture dishes
(P35G-101; MatTek Corp., Ashland, MA). Confluent cells between
the 10th and 20th passages were used for these experiments. Prior
to each experiment, the medium was switched to serum-free DME
and the cells were incubated for 24 h. Then, a cytokine cocktail
containing final concentrations of 25 units/ mL IL-1â, 30 ng/ mL
TNF-R, 150 units/ mL IFN-γ, and 12.5 µg/ mL LPS was added to
the medium. After another 12-h incubation, the activated cells
were used for the experiment.
Figure 2. Excitation (A) and emission (B) spectra for DAF-2 at 37
°C in 0.1 M sodium phosphate buffer (pH 7.4) with NO values ranging
from 0 to 2.4 µM. NO solution was added to 10 µM DAF-2 solution
under aerobic conditions. The spectra were obtained from an average
of seven accumulations 15 min after the addition of NO solution.
an MCID (Imaging Research Inc., St. Catharines, ON, Canada),
which is an imaging system including a charge-coupled device
(CCD) camera. Images were taken at intervals of 30 s. A confocal
laser scanning microscope (Noran Instruments Inc., Middleton,
WI) equipped with an objective lens (×40) and a long-pass
emission filter (515 nm) was used for scanning cells in 2-µm
sections from the bottom.
Imaging P rocedures. The cells were incubated for 1 h at
37 °C in KRP containing 10 µM DAF-2 DA (0.2% DMSO was used
as a cosolvent) for loading, washed with KRP, and placed in KRP
containing L-Arg or L-NMMA. They were mounted on an inverted
fluorescence microscope (IX70; Olympus, Tokyo, Japan) equipped
with an objective lens (×20), an excitation filter (490 nm), a
dichroic mirror (505 nm), and a long-pass emission filter (515 nm).
The temperature of the stage was maintained at 37 °C with a
microwarm plate (DC-MP10DM; Kitazato, Shizuoka, Japan). Light
exposure time was 132 ms. Optical signals were recorded with
RESULTS AND DISCUSSION
Chemical P roperties of DAFs. Diaminofluoresceins (Figure
1) were obtained according to the reaction scheme shown.
Reduction of aminonitrofluoresceins by catalytic hydrogenation
or with acid and metal combinations proved unsatisfactory, so we
employed an alkaline reduction system with sulfide and hydro-
sulfide ion.22
(26) Nishikawa, Y.; Hiraki, K. Analytical Methods of Fluorescence and Phospho-
rescence; Kyoritsu Publishing Co.: Tokyo, 1984; pp 76-80.
2450 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

Figure 3. Time course of fluorescence intensity of DAF-2 depending
on NO generated from NONOates. Varying concentrations of two
NONOates with different half-lives were incubated in 2-mL reaction
volumes containing 0.1 M sodium phosphate buffer (pH 7.4) and 10
µM DAF-2 at 37 °C. The fluorescence intensity was determined at
515 nm with excitation at 495 nm. (A) NOC 12 was added immediately
after it had been dissolved in 0.1 M sodium phosphate buffer (pH
7.4). (B) NOC 13 was added. Final concentrations: (1) 5 (2) 50, (3)
100, and (4) 500 µM. aHalf-lives at 22 °C are taken from the
literature.24
Figure 5. Detection of triazole formation by endotoxin-stimulated
macrophages using reversed-phase HPLC. Fluorescence was moni-
tored at an excitation wavelength of 495 nm and an emission
wavelength of 515 nm. HPLC conditions are given the Experimental
Section. (A) Standard DAF-2. (B) Endotoxin-stimulated RAW 264.7
cells were incubated with DAF-2 for 2 h. Endotoxins were LPS (10
ng/mL) and IFN-γ (10 units/mL). (C) Nonstimulated RAW 264.7 cells
were incubated with DAF-2 for 2 h.
Figure 4. Effect of pH on the fluorescence intensity of DAF-2 T
and DAF-5 T (1 µM) at 20 °C. Triazole forms of DAF-2 and DAF-5
(final 1 µM) were added to sodium phosphate solution (78 mM)
adjusted to various pH values. The pH was measured after mixing.
The fluorescence intensities of DAF-2 T and DAF-5 T were deter-
mined at 515 and 520 nm with excitation at 495 and 505 nm,
respectively. The curves were fitted with the following equations:
(DAF-2 T) intensity ) 460/(1 + 106.27-pH) - 55.6/(1 + 107.94-pH), R
) 0.9997; (DAF-5 T) intensity ) 505/(1 + 104.59-pH) - 119/(1 +
107.41-pH), R ) 0.9996.
Figure 6. Schematic representation of dye behavior in the cell.
DAF-2 DA permeates through the cell membrane and is hydrolyzed
to yield DAF-2, which is retained in the cell owing to its relatively poor
permeating ability. Loaded DAF-2 reacts with NO to form fluorescent
DAF-2 T.
Although the mechanism of fluorescence of the triazole form
compounds is not entirely clear, one possibility is as follows. Minta
et al. proposed for fluorescent xanthene derivatives that electron
donation from the amino nitrogen to the phthalic ring and then
to the xanthene would increase the double-bond character
between the phthalic ring and the xanthene, resulting in hindered
planarity of the xanthene and radiation deactivation of fluores-
cence.9 Although the structure in the excited state rather than
in the ground state must be taken into consideration because the
absorption spectra of the diamino forms and triazole forms are
similar, it can be qualitatively assumed that fluorescence disap-
pears when the functional groups donate electrons to the phthalic
ring by resonance. Thus, the fluorescence intensity of fluorescein
derivatives can be modified by functional group substitution with
electron-donating or -withdrawing groups.
The absorbance and fluorescence properties for DAF-1-DAF-6
are summarized in Table 1. Conversion of DAFs to the triazole
form by reaction with NO caused little change of the absorbance
maximums, but greatly increased the quantum efficiency. The
excitation and emission wavelengths of DAF-4-DAF-6, with a
chlorine atom adjacent to phenolic OH, were shifted to longer
wavelengths than those of DAF-1-DAF-3. DAFs possess favor-
able properties for cellular imaging applications; namely, DAF has
a visible excitation wavelength, which is less damaging to cells,
and is not subject to interference from the autofluorescence of
biological samples. For example, Figure 2 shows the excitation
and emission spectra of DAF-2. The increase of fluorescence
intensity was dependent on the concentration of NO. Figure 3
shows the time course of fluorescence intensity of DAF-2 after
the addition of NO-releasing compounds (NOCs).24 The intensity
increased according to the half-life and concentration of added
NOC.
Judging from NO standard curves calibrated with 10 µM DAFs,
DAF-2 and DAF-4 showed a little higher sensitivity than the others.
The detection limit of NO by DAF-2 was 5 nM.
Analytical Chemistry, Vol. 70, No. 13, July 1, 1998 2451

Figure 7. Bright-field and fluorescence images of the same group of activated rat aortic smooth muscle cells loaded with DAF-2 DA in the
presence of L-Arg (1 mM). These images are shown in pseudocolor and correspond to the fluorescence intensity data in Figure 8A. Instrument
settings were similar to those described in Figure 8.
The fluorescence intensity of fluorescein with two phenolic OH
groups is almost completely quenched when the phenolic OH
group is protonated. DAFs exhibit similar properties. Figure 4
shows the pH profile of fluorescence intensity of the triazole forms
of DAF-2 and DAF-5 (DAF-2 T, DAF-5 T). The fluorescence of
DAF-2 T strikingly decreased below pH 7. The titration curve
fitted a pKa of 6.27 ( 0.02. Conversely, it was found that DAF-
1-DAF-3 were useful in media above pH 7. In fact, we had
designed and synthesized DAF-4-DAF-6 with the electron-
withdrawing chlorine substituent to lower the pKa value with the
aim of obtaining a stable fluorescence intensity at physiological
pH. Although the electron-withdrawing effect of the chlorine atom
did lower the pKa values of DAFs, as shown in Figure 4, DAF-5 T
(pKa, 4.59 ( 0.01), there was a reduction of their fluorescence
intensity from pH 7 to 9, probably due to deprotonation of the
triazole moiety (pKa, 7.94 ( 0.17 for DAF-2 T; pKa, 7.41 ( 0.04
for DAF-5 T). It was reported that the pKa of benzotriazole was
8.2 and that of 5-chlorobenzotriazole was 7.7.28,29 The protonation
of phenolic OH significantly reduced the fluorescence intensity
because of the blue shift of the absorption wavelength owing to
hindrance of electron delocalization, whereas deprotonation of the
triazole ring slightly decreased the intensity because of the small
electron donation to the conjugated double bonds. The chlorine
substitution, lowering the pKa value of phenolic OH, resulted in
unstable fluorescence intensity around physiological pH. Taking
these effects into consideration, we concluded that DAF-2 would
be most appropriate for detecting NO in neutral pH buffers.
The reaction mechanisms of NO and DAF are complex
because DAFs react not with NO itself but with an active
intermediate formed in the course of the oxygen oxidation of NO
-
to NO2
. N2O3 is formed as shown in eq 2 and is a potent
nitrosating reagent. The rate constant for eq 2 (1.1 × 109 M-1
30
s-1
)
indicates that N2O3 is immediately formed when NO is
•
oxidized to NO2. The oxidizing reaction of N2O3 is competitive
with hydrolysis to NO2
-
. We found that the yield of triazolo-
fluorescein based on NO was 18% in neutral solution in the
absence of biological samples. Since it was reported that the rate
constant for nitrosation of morpholine by N2O3 was 6.4 × 107 M-1
s-1 31
, this step cannot be rate-determining. The rate-determining
step is probably the formation of •NO2 from NO (eq 1). The third-
order rate constant for reaction of two NO and one oxygen
(27) Ross, R.; Glomset, J.; Kariya, B.; Harker, L. Proc. Natl. Acad. Sci. U.S.A.
1 9 7 4 , 71, 1207-1210.
(30) Gr¨atzel, M.; Taniguchi, S.; Henglein, A. Ber. Bunsen-Ges. Phys. Chem. 19 70,
(28) Fagel, J. E., Jr.; Ewing, G. W. J. Am. Chem. Soc. 1 9 5 1 , 73, 4360-4362.
(29) Katritzky, A. R.; Rees, C. W. Comprehensive Heterocyclic Chemistry; Perga-
mon Press: Oxford, 1984; Vol. 5, p 690.
74, 488-492.
(31) Lewis, R. S.; Tannenbaum, S. R.; Deen, W. M. J. Am. Chem. Soc. 1 9 9 5 ,
117, 3933-3939.
2452 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

molecule was reported by Kharitonov et al. to be 6.3 ( 106 M-2
s-1 32
Due to the slow reaction speed of NO oxidation, the
.
chemical yield of the product was low. This result has two
implications. The first is that the detection limit could be higher
if the reaction speed were faster; so in other words, the rate at
which NO is oxidized imposes an intrinsic limitation on detection
by this method. The other point is that this detection reagent
does not react directly with NO, but reacts with the oxidized form
of NO. So, the measurement should not interfere too much with
the signal transduction by NO. Nevertheless, the specificity for
NO is high, because DAFs did not react in neutral solution with
-
mobile and stable oxidized forms of NO, such as NO2- and NO3
,
Figure 8. Fluorescence response of DAF-2 DA-loaded rat aortic
smooth muscle cells, reflecting NO production. Smooth muscle cells,
12 h after LPS and cytokine activation, were incubated with 10 µM
DAF-2 DA for 1 h at 37 °C for dye loading. Then, the cells were
washed with KRP, and the medium was changed to KRP containing
L-arginine (L-Arg) or N-monomethyl-L-arginine (L-NMMA) 10 min
before time 0. Fluorescence excited at 490 nm (515-nm emission)
was measured at 30-s intervals. (A) Fluorescence intensity of
activated cells obtained when L-Arg (1 mM) was present in the
medium. (B) Fluorescence intensity of activated cells in the presence
of L-Arg (1 mM) and L-NMMA (10 mM). (C) Fluorescence intensity of
inactivated cells in the presence of L-Arg (1 mM). Lines show mean
values of the average fluorescence intensity in areas containing a
cell. The zero point fluorescence intensity was taken as the initial
intensity at the start of measurement.
and other reactive oxygen species, such as O2•-, H2O2, and
ONOO-, to yield any fluorescent product. Under physiological
conditions, triazolofluorecein is not formed in the absence of NO.
Detection of NO P roduction by Macrophages. Isocratic
elution with 10 mM sodium phosphate buffer pH 7.4/ acetonitrile
(94:6 v/ v) on a reversed-phase column separated DAF-2 and
DAF-2 T. Judging from the determination by HPLC with UV-
visible detector, the retention times (rt) of DAF-2 and DAF-2 T
were 4.81 and 4.23 min, respectively. Figure 5 shows the triazole
formation evaluated by HPLC with a fluorescence detector.
Standard DAF-2 shows little fluorescence at rt 4.81 min (Figure
5A). The supernatant of cultured macrophages (RAW 264.7)
showed marked fluorescence at rt 4.23 min, and this was
augmented after endotoxin stimulation (Figure 5B). The non-
stimulated cells produced a smaller amount of the triazole form
(Figure 5C). This result reflects substantial NO production by
macrophages even without LPS and IFN-γ stimulation.
NO Imaging. We designed and synthesized DAF-2 DA
(Figures 1 and 6) for imaging of NO and confirmed that it
permeated well into the cells, where it was quickly transformed
into DAF-2 by esterase in the cytosol. The hydrolysis was
confirmed by HPLC detection. DAF-2 DA (10 µM) was hydro-
lyzed quickly within 10 min by the cytosolic solution of cell lysate
(data not shown). During the acetylation of the phenolic OH of
DAF-2, amino groups might also be acetylated. Therefore,
phenolic OH was deprotonated over cesium carbonate and
acetylation was carried out carefully with acetic anhydride (see
Figure 1). This procedure avoided the acetylation of the amines.
The dye was applied to the imaging of NO in cultured rat aortic
smooth muscle cells by using a fluorescence microscope equipped
with fluorescence filters for fluorescein chromophores. The cells
were incubated with 10 µM DAF-2 DA for 1 h at 37 °C for dye
loading. Fluorescein is easily photobleached by strong excitation
light. Photobleaching was observed with DAF-2, but reduction
of the light exposure time and the lower excitation frequency
reduced this effect to a manageable level. Confocal laser scanning
microscopy indicated that fluorescence was emitted from the
whole cell body, including the nucleus (data not shown). This
implies that DAF-2 regenerated intracellularly is distributed
throughout the whole cell. There was no indication of any cell
damage caused by loading the dye. Figure 7 shows the bright-
field and fluorescence images, corresponding to the fluorescence
intensity data of Figure 8A. Figures 7 and 8A show that the
fluorescence intensity in endotoxin- and cytokine-activated cells
increased owing to DAF-2 T production from DAF-2 by reaction
with NO. The increase was suppressed by the NO synthase
inhibitor L-NMMA (Figure 8B). The initial increase of fluores-
cence resulted from NO generated before the inhibition of NO
synthase. Figure 8C shows that the fluorescence did not increase
in inactivated cells, which did not produce NO. When an NO
donor (NONOate, 1 mM NOC13) was added to the medium of
inactivated cells loaded with DAF-2 DA, the fluoresence intensity
in the cells was suddenly increased (data not shown).
We determined NO evolved in the supernatant by the addition
of the nonesterified indicator to the medium and by the measure-
ment of the triazole formation with a fluorescence spectropho-
tometer. As a result, it was estimated that NO was produced at
the rate of 2 fmol/ h by a single cell under these conditions. It is
highly likely that the concentration of NO in the cell before
diffusion is considerably higher than that in the medium thus
examined. Real-time visualization of the production and diffusion
of NO with our new fluorescent indicators is useful for direct
analysis of the cellular functions of NO.
ACKNOWLEDGMENT
The authors are indebted to Dr. Y. Imai and Prof. T. Irimura
for donating macrophages, and Dr. N. Hirashima for operating
the confocal laser scanning microscope. This work was supported
by a Grant-in-Aid for Scientific Research on Priority Areas No.
09273216 from the Ministry of Education, Science, Sport and
Culture of Japan.
Received for review February 13, 1998. Accepted April 20,
1998.
(32) Kharitonov, V. G.; Sundquist, A. R.; Sharma, V. S. J. Biol. Chem. 1 9 9 4 ,
269, 5881-5883.
AC9801723
Analytical Chemistry, Vol. 70, No. 13, July 1, 1998 2453