Inhibitory effects of Mannich bases of heterocyclic chalcones on NO
production by activated RAW 264.7 macrophages and superoxide anion
generation and elastase release by activated human neutrophils
M. Vijaya Bhaskar Reddy
a, Tsong-Long Hwang
b,, Yann-Lii Leu
b, Wen-Fei Chiou
c, Tian-Shung Wu
a,d,⇑ aDepartment of Chemistry, National Cheng Kung University, Tainan 701, Taiwan, ROCbGraduate Institute of Natural Products, College of Medicine, Chang Gung University, 259 Taoyan, Taiwan, ROC c
National Research Institute of Chinese Medicine, Taipei 112, Taiwan, ROC
d
College of Pharmacy and Chinese Medicine Research and Development Center, China Medical University, Taichung, Taiwan, ROC
a r t i c l e
i n f o
Article history:
Received 11 January 2011 Revised 17 February 2011 Accepted 18 February 2011 Available online 8 March 2011 Keywords:
Mannich bases of heterocyclic chalcone analogs NO production inhibition (O2) generation Elastase release [Ca2+ ]imobilization
a b s t r a c t
Some chalcones exert potent anti-inflammatory activities. Mannich bases of heterocyclic chalcones inhibited nitric oxide (NO) production in lipopolysaccharide and interferon-cstimulated RAW 264.7 macrophages. Also Formyl-Met-Leu-Phe and cytochalasin B induced superoxide anion generation (O2)
and elastase release in human neutrophils. Mannich bases of heterocyclic chalcone analogs exhibited potent inhibitory effects on NO production with IC50values ranges between 10.5 and 0.018lM, O2
gen-eration (IC5039.87–0.68lM) and elastase release (IC5039.74–0.95lM). Compound 29 (IC500.055lM)
and 34 (IC500.018lM) were showed excellent inhibition on NO production. On the other hand,
com-pounds 2 and 8 showed potent inhibition on O2generation and elastase release. Therefore, these four
compounds may be new leads for development of anti-inflammatory activities. The structure–activity relationships are also discussed.
Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
1. Introduction
Many laboratories and clinical studies evidenced that oxidative stress imposed by reactive oxygen species (ROS) plays a crucial role in the pathophysiology associated with atherosclerosis, neurode-gradative diseases and carcinogenesis.1Oxidative stress developed
when there is an imbalance between the generations of ROS and their removal, resulting in potential cell damages. ROS species in-clude free radicals such as hydroxyl radicals, peroxy radicals and superoxide anion radicals; and other reactive species like hydrogen peroxide, singlet oxygen and nitric oxide (NO). Among these ROS species, nitric oxide (NO) is a biologically active and paramagnetic free radical, which it has a reactive short half-life.2,3 NO is an
important mediator involved in the regulation of many physiolog-ical and pathologphysiolog-ical processes including neurotransmission and smooth muscle relaxation. The free radical nature of NO and high reactivity of superoxide (O2), with subsequent generation of
ONOO, renders NO to a potent pro-oxidant molecule inducing
po-tential oxidative damages toward cellular targets.4,5The formation
of NO is catalyzed by the enzyme nitric oxide synthase (NOS) via the NADPH- and O2-dependent oxidation of L-arginine.6 Three
distinct isoforms of NOS have been identified including constitu-tive endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS). The endothelial and neuronal NOS are predominantly presented in the vascular endothelium and nervous system, respectively. The iNOS generate high levels of NO that modulates inflammations through multiple pathways and plays an important role in the regulation of immune reactions.7,8Overproduction of
NO by iNOS implicates a number of diverse physiological and path-ological processes, such as vasodilation, nonspecific host defense, ischemia, reperfusion injury, chronic or acute inflammation, rheu-matoid arthritis and onset of colitis.9A variety of exogenous
stim-uli, including lipopolysaccharide (LPS), interferon gamma (INF
c
), tumor necrosis factor (TNFa
), other induced ultraviolet radiation and ozone,10,11stimulate the expression of iNOS.Human neutrophils are known to play important roles in the host defense against microorganisms. They are also critical in the pathogenesis of various diseases such as rheumatoid arthritis, ischemia, reperfusion injury, chronic obstructive pulmonary dis-ease (COPD) and asthma.12–16In response to the diverse stimuli,
activated neutrophils secrete series of cytotoxins, such as superox-ide anion (O2), granule proteases, and bioactive lipids. Despite
this, currently there are only a few available agents that directly modulate neutrophils’ pro-inflammatory responses in clinical practices. Therefore, the above mentioned inhibitory effects are
0968-0896/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2011.02.038
⇑ Corresponding author. Tel.: +886 6 2747538; fax: +886 2 2740552. E-mail address:[email protected](T.-S. Wu).
This author has the same contribution as the first author.
Contents lists available atScienceDirect
Bioorganic & Medicinal Chemistry
the important therapeutic targets for potential anti-inflammatory diseases.
Mannich bases of chalcones and related compounds were dis-played significant cytotoxicity towards murine P388 and L1210 leukemia cancer cell lines,17 as well as a number of other tumor
cell lines. Previously, we reported that the synthesis and biological evaluations of Mannich bases of heterocyclic chalcone analogs
(1–39) (Scheme 1) are cytotoxic agents against four human cancer cell lines including PC-3 (prostate cancer), MCF-7 (human breast cancer), KB (nasopharyngeal carcinoma) and KB-VIN (vincristine-resistant KB subline).18To the best of our knowledge,
there is no existing literatures focused on the inhibitory effects of NO production, O2 generation and elastase release by Mannich
bases of heterocyclic chalcones. In continuation of our interests
RO OH N O O B ring R B-ring 1. H 2. Me 3. isopropyl 4. prenyl 2-pyridyl 2-pyridyl 2-thiophene 3-methyl-2-thiophene RO OH O B ring N O R B-ring 5.ethyl 3-pyridyl 6. ethyl 2-furan 7. ethyl 5-methyl-2-furan 8. ethyl 2-pyridyl 9.ethyl phenyl 10.ethyl 3-methyl-2-thiophene 11.methyl 3-pyridyl 12.methyl phenyl 13.methyl 4-CH3OC6H4 14.methyl 2-thiophene 15.methyl 2-furan 16.isopropyl 2-thiophene HO N O O B ring B-ring 17. 18. 19. 20. 21. 22. 23. 24. 2-pyridyl 3-pyridyl 4-pyridyl phenyl 2-furan 2-thiophene 3-methyl-2-thiophene 5-methyl-2-furan HO N O O B ring N O B-ring 25. 2-pyridyl 26. 3-methyl-2-thiophene O Bring OH N O B-ring 27. 2-pyridyl 28. 3-pyridyl 29. 4-pyridyl O OH N O N N O 34. O B ring OH N O B-ring 30. 31. 32. 33. 2-furan 2-thiophene 3-methyl-2-thiophene phenyl A ring O OMe OH N A-ring 37. 38. 39. 2-thiophene 2-furan 3-pyridyl A ring O OMe OH N O A-ring 35. 36. 36H4-Me-C4-Me-C6H4 Scheme 1.
on Mannich bases of heterocyclic chalcone analogs, herein we describe the inhibition of NO production in LPS plus INF-
c
activated RAW 264.7 macrophages in a preliminary in vitro test. Also cytotoxicity was evaluated by MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Most of the compounds showed potent inhibition of NO production with IC50values rangesbetween 10.5 and 0.018
l
M comparing to the reference compound, AMG (amtolmetin guacyl) (IC5051.30l
M).In addition, we examined the inhibitory effects on Formyl-Met-Leu-Phe (FMLP) and cytochalasin B (CB) stimulated O2generation
and elastase release in human neutrophils. The IC50values ranges
between 0.68 and 39.87
l
M and 0.95–39.74l
M, respectively. Among 39 compounds, 29 and 34 showed potent NO production inhibitions in LPS plus INF-c
activated RAW 264.7 macrophages with IC50values of 0.055 and 0.018l
M, respectively. Compounds2 and 8 showed potent inhibitory effects on FMLP/CB stimulated O2generation and elastase release in human neutrophils. In the
further study, we evaluated compounds 2 and 21 for FMLP-acti-vated increasing [Ca2+]
imobilization in human neutrophils.
2. Results and discussions
To clarify the structure–activity relationships (SAR) of Man-nich bases of heterocyclic chalcones 1–39, the inhibition of NO production in LPS plus INF-
c
activated RAW 264.7 macrophages, O2 generation, and elastase release in FMLP/CB stimulatedhu-man neutrophils. Compounds 1 and 2 are 2-pyridyl group in ring-B; and hydroxyl and methoxyl groups at C-40 position in
ring-A, respectively. They showed equipotent inhibition of NO production in RAW 264.7 macrophages with an IC50 value of
1.7
l
M. But 2 exhibited more potent inhibitory effects on FMLP/CB induced O2 generation and elastase release with IC50
values of 0.68 and 0.95
l
M than 1(O2 IC50 12.64l
M andelas-tase release IC50of 21.17
l
M). These results indicate thatmeth-oxy group at C-40 position in which 2 is responsible for the
enhancement of inhibitory effects on O2 generation and
elas-tase release in human neutrophils.
Analogs 3 and 16 are positional isomers, which have morpho-line Mannich base group at C-30 position and C-50, respectively.
Compound 3 (IC502.21
l
M) showed less inhibited NO productionthan 16 (IC500.23
l
M). Also turn 3 (IC50 3.39l
M) showed morepotent on O2 generation than 16 (IC
5019.39
l
M). These resultsclearly indicate bulky Mannich base group at C-30position in 3 is
weak in responding to inhibit NO production in RAW 264.7 cells. In addition, compound 4 (IC50 0.93
l
M) had prenyloxy group atC-40 and 3-methyl-2-thiophene moiety as ring-B, showed more
potent NO production inhibition than 3 (IC502.21
l
M).Compounds 5–8 are 3-pyridyl, 2-furan, 5-methyl-2-furan and 2-pyridyl substitution pattern in ring-B, respectively, showed significant inhibition on NO production with IC50values ranges
be-tween 10.50 and 16.50
l
M. But with 3- and 2-pyridyl substitution in ring-B of compounds 5 and 8 exhibited more potent inhibition on O2 generation in FMLP/CB induced human neutrophils withIC50values of 2.42 and 0.89
l
M, respectively. This indicated thatpyridine ring was probably responsible for the enhancement of inhibition on O2 generation and elastase release in human
neutrophils.
The un-substituted phenyl group in B-ring, ethoxyl and meth-oxyl groups at C-40 in 9 (IC
50 0.29
l
M) and 12 (IC50 0.20l
M),respectively, showed potent NO production inhibition in LPS plus IFN-
c
induced RAW 264.7 macrophage cells.Compound 14 (IC503.61
l
M) showed significantly inhibited NOproduction, when methoxyl group was replaced with isopropoxyl group at C-40 position in ring-A, higher inhibition was observed
in 16 (IC500.23
l
M). So isopropoxyl group at C-40 position maybe responsible for the enhancement of NO production inhibition in 16. However, the reverse results were observed in the inhibition of O2generation for compounds 14 (IC5010.19
l
M) and 16 (IC5019.39
l
M).Based on the in vitro preliminary results for compounds 5–16, 3-pyridyl group as ring-B analogs (5 and 11), 2-pyridyl (8), phenyl (9 and 12) and 4-methoxyphenyl (13) showed more potent inhibitory effects in FMLP/CB induced O2 generation than five
membered heteroatomic ring-B substituted analogs (6, 7, 10 and 14–16).
Compounds 17, 18 and 19 are 2, 3 and 4-pyridyl groups in ring-B, respectively. Among these, 18 (IC50 0.82
l
M) showed morepotent NO production inhibition than 17 (IC50 3.41
l
M) and 19(IC50 4.14
l
M). When the pyridyl moiety as ring-B was replacedwith phenyl moiety, drastically decreased NO production inhibi-tion was observed in compound 20 (IC5015.60
l
M).Compounds 21 (IC500.16
l
M) and 22 (IC500.7l
M) are 2-furanand 2-thiophene groups as ring-B, respectively, were more po-tently inhibited NO production than methyl substituted analogs 23 (IC501.39
l
M) and 24 (IC501.28l
M).Analogs 27–29 had 2, 3 and 4-pyridyl moieties in ring-B, respec-tively, and hydroxyl and morpholine Mannich base groups at C-30
and C-40positions, showed more potent inhibition on NO
produc-tion in LPS plus INF-
c
activated RAW 264.7 macrophages than the isomers 17–19. The similar results were observed in FMLP/CB in-duced O2 generation, elastase release, and inhibition in humanneutrophils for compounds 27–29. These results clearly indicate that C-40hydroxyl group has conjugation with carbonyl carbon in
17–19, probably decreasing the inhibitory effects in both NO pro-duction in RAW 264.7 macrophage cell lines, O2 generation and
elastase release in human neutrophils.
Compounds 25 (IC502.52
l
M) and 26 (IC500.44l
M) containedtwo Mannich base groups at C-30and C-50positions, showed more
potent inhibitors of NO production than corresponding mono substituted Mannich base analogs 17 (IC503.41
l
M) and 23 (IC501.39
l
M). Analogs 30–33 significantly inhibited NO production with IC50 values ranges between 2.30 and 3.39l
M. Compound34 (IC500.018
l
M) showed more potent inhibition of NOproduc-tion than corresponding isomer 25 (IC502.52
l
M).Compounds 21–26 and 30–32 have five membered heteroatom-ic substitution as ring-B, exhibited less inhibitory effects on both O2 generation (IC50 27.38–9.60
l
M) and elastase release (IC5039.74 to 15.40
l
M) in FMLP/CB induced human neutrophils. Compound 34 with two Mannich base groups at C-20and C-40and hydroxyl group at C-30in ring-A, exhibited more potent
inhib-itory effects in FMLP/CB stimulated O2generation (IC
501.66
l
M),elastase release (IC50 1.66
l
M) in human neutrophils, andinhibition of NO production (IC500.018
l
M) than 25, which haveMannich base groups at C-30and C-50and hydroxyl at C-40position.
The results indicate that hydroxyl group at C-40 in ring-A was
probably responsible for less inhibitory effects observed in all cases, due to hydroxyl group’s conjugation with carbonyl carbon. The methyl substituted analogs 35 and 36 exhibited significant inhibitory effects on FMLP/CB stimulated O2generation and
elas-tase release in human neutrophils (Tables 1–3).
3. Conclusions
In summary, a series of Mannich bases of heterocyclic chalcone analogs (1–39) were evaluated for their inhibition on NO produc-tion in activated RAW 264.7 macrophages, FMLP/CB induced O2
generation, and elastase release in human neutrophils. Compounds 29 (IC500.055
l
M) and 34 (0.018l
M) exhibited potent inhibitionon NO production in activated RAW 264.7 macrophages. The ef-fects of compounds (1–34) on cell viability were determined by
MTT assay in RAW 264.7 macrophages. No significant cytotoxicity was observed for compounds 29 (IC50110
l
M) and 34 (24.30l
M)in RAW 264.7 macrophages. These data revealed that the inhibi-tory effect of compounds 29 and 34 on NO production at concen-trations which were well below their cytotoxicity. However, some compounds (5, 6, 12, 17, and 20) had similar inhibitory po-tency for NO production and cytotoxicity. Therefore, we could not rule out the possibility that these compounds inhibited NO production due to their cytotoxicity in RAW 264.7 macrophages.
In addition, compounds 2 and 8 were potent inhibitors of FMLP/ CB induced O2 generation and elastase release in neutrophils.
Further study showed that significant inhibition on FMLP-induced increase in [Ca2+]
imobilization by compounds 2 was observed in
human neutrophils. In contrast, 21 failed to change the [Ca2+]i
mobilization of human neutrophils caused by FMLP. The bacterial peptide FMLP is the first to be identified and is a highly potent leu-kocyte chemoattractant. FMLP activates neutrophils by binding to the G protein-coupled receptor (GPCR) on the membrane. Stimula-tion of GPCR induces the Ca2+signal via activation of phospholipase
C, which hydrolyzes phosphatidylinositol 4,5-bisphosphate into inositol trisphosphate and diacylglycerol, resulting in an increase in [Ca2+]
iand activation of PKC, respectively.19Many cellular
func-tions of neutrophils, such as respiratory burst and degranulation, are regulated by the Ca2+signals.20The magnitude and duration
of [Ca2+]
i signal responses to GPCR are obviously important.
In-creases in [Ca2+]
ihave profound effects on neutrophils, including
the initiation of cytoskeletal changes, degranulation, and respira-tory burst.20,21Our results suggest that the inhibition of human
neutrophil O2generation and elastase release by compound 2 is
through attenuation of calcium signaling pathway (Fig. 1).
Table 2
Effects of compounds on superoxide anion generation and elastase release by human neutrophils in response to FMLP/CB
Compound Superoxide anion IC50(lM)aor (Inh%) Elastase IC50(lM)a or (Inh%) 1 12.64 ± 0.36 21.17 ± 2.91 2 0.68 ± 0.05 0.95 ± 0.04 3 3.39 ± 0.22 NTb 4 (21.54 ± 3.41)⁄⁄ NTc 5 2.42 ± 0.37 3.48 ± 0.29 6 (39.87 ± 4.67)⁄⁄ NTc 7 19.95 ± 1.29 NTc 8 0.89 ± 0.07 1.24 ± 0.16 9 3.35 ± 0.24 NTc 10 19.95 ± 1.29 NTc 11 2.65 ± 0.17 4.52 ± 0.60 12 2.86 ± 0.06 NTc 13 5.77 ± 0.80 NTc 14 10.19 ± 0.92 NTc 15 15.81 ± 1.49 (17.92 ± 2.05)⁄⁄ 16 19.39 ± 3.82 NTc 17 15.17 ± 0.65 20.85 ± 2.54 18 23.52 ± 2.74 (20.38 ± 5.69)⁄ 19 8.85 ± 0.47 13.65 ± 1.19 20 16.57 ± 1.44 (9.32 ± 2.41)⁄ 21 (27.38 ± 4.00)⁄⁄ (16.17 ± 6.00) 22 25.39 ± 2.24 (19.29 ± 5.58)⁄⁄ 23 16.40 ± 3.43 18.20 ± 1.54 24 25.57 ± 1.81 (13.50 ± 5.52) 25 14.77 ± 0.68 18.89 ± 4.18 26 (33.78 ± 2,61)⁄⁄⁄ (15.40 ± 4.79)⁄ 27 2.63 ± 0.11 2.47 ± 0.67 28 5.96 ± 1.15 6.66 ± 0.82 29 1.60 ± 0.18 2.28 ± 0.42 30 18.19 ± 1.16 (39.74 ± 4.48)⁄⁄⁄ 31 10.48 ± 1.06 24.31 ± 1.40 32 9.60 ± 1.77 18.61 ± 2.92 33 2.81 ± 0.43 5.19 ± 1.06 34 1.66 ± 0.43 1.44 ± 0.13 35 3.16 ± 0.03 2.95 ± 0.80 36 2.76 ± 0.16 6.97 ± 1.77 37 8.32 ± 0.82 NTc 38 4.18 ± 0.26 NTc 39 13.03 ± 1.73 20.08 ± 2.43 DPId 1.02 ± 0.35 NT PMSFd NT 95.0 ± 25
Percentage of inhibition (Inh%) at 30lM concentration. Results are presented as mean ± S.E.M. (n = 3–4).⁄
P <0.05,⁄⁄
P <0.01,⁄⁄⁄
P <0.001 compared with the control value.
a
Concentration necessary for 50% inhibition (IC50). b
Compound 25 reacted with substrate.
c
Compounds 4, 6, 7, 9, 10, 12–14, 16, 37 and 38 had a strong absorbance at 405 nm.
dDiphenyleneiodonium (DPI, a NADPH oxidase inhibitor) and
phen-ylmethylsulfonylfluoride (PMSF, a serine protease inhibitor) were used as the positive controls in the generation of superoxide anion and elastase release, respectively.
Table 1
Nitric oxide production and cytotoxic effects of compounds 1–34 in LPS plus IFN-c
induced RAW 264.7 macrophages
Compound NO inhibition IC50(lM) MTT assay IC50(lM)
1 1.73 11.40 2 1.73 10.80 3 2.21 34.00 4 0.93 11.00 5 15.40 4.46 6 16.50 16.90 7 10.50 91.30 8 15.10 0.18 9 0.29 1.60 10 8.13 2060.00 11 11.80 2810.00 12 0.20 0.19 13 3.35 1860.00 14 3.61 148.00 15 6.06 1440.00 16 0.23 47.70 17 3.41 6.56 18 0.82 53.20 19 4.14 111.00 20 15.60 35.00 21 0.16 10.00 22 0.47 6830.00 23 1.39 NDa 24 1.28 0.006 25 2.52 190.00 26 0.44 56.90 27 0.72 78.00 28 0.23 ND 29 0.055 110.00 30 2.50 17.20 31 2.49 12.70 32 3.39 182.00 33 2.30 9.95 34 0.018 24.30 AMG 51.30 ND aNot determined. Table 3
Effects of compounds 2 and 21, on the peak [Ca2+]
iand the time taken for this
concentration to decline to half of its peak value (t1/2) in FMLP-activated neutrophils
Drugs Peak [Ca2+
]i
values (nM)
Time taken to decline to half peak values (t1/2) (s)
DMSO 280.75 ± 8.91 31.47 ± 3.90 2 (10lM) 208.65 ± 15.35⁄
8.43 ± 1.42⁄⁄
21 (10lM) 276.51 ± 6.79 28.10 ± 2.11
Neutrophils labeled with fluo-3AM as described under Section4were stimulated with 0.1lM FMLP in the presence of 1 mM Ca2+
, and fluorescence was monitored at 37 °C with stirring. All data are expressed as mean ± SEM.⁄
P <0.05,⁄⁄
P <0.01, compared to the control value.
4. Materials and methods
4.1. Mannich bases of heterocyclic chalcone analogs
Thirty nine Mannich bases of heterocyclic derivatives (1–39) were synthesized as described previously,18 and their chemical
structures are shown inScheme 1. The structures were confirmed by IR,1H,13C NMR, EIMS, HREIMS and elemental analysis. 4.2. Cell culture
The murine macrophages cell line, RAW 264.7 cells, was obtained from the American Type Culture Collection (ATCC, TIB 71, Rockville, MD). Cells were cultured in 75 cm2plastic flasks (Corning–Costar)
with Dulbecco’s modified Eagle’s medium (DMEM), supplemented with antibiotics (100 U mL1penicillin A and 100
l
g mL1ofstrep-tomycin), and 10% heat-inactivated fetal calf serum (FCS), and main-tained in a 37 °C humidified incubator containing 5% CO2in air for
1 h. Non-adherent cells were removed by various pipetting after centrifugation, and adherent cells were cultured in 96-well plates with 200
l
L of cultured medium reached until confluence (approx-imately 200,000 cells per well) followed by adding fresh medium containing LPS (1l
g mL1) and IFN-c
(50 U mL1).4.3. Nitrite measurement
The RAW 264.7 cells were cultured in order to induce iNOS, fresh cultured medium containing LPS (1
l
g mL1) and IFN-c
(50 U mL1) was added. Nitrite accumulation in the medium was
measured at 24 h with the application of LPS (1
l
g mL1) plusIFN-
c
(50 U mL1). To assay the effect of drugs on nitriteproduc-tion, compounds were added together with LPS/IFN-
c
. Nitrite was measured by adding 100l
L of Griess reagent (1% sulfanil-amide and 0.1% naphthylenediamine in 5% phosphoric acid) to 100l
L samples of medium. The optical density of 550 nm (OD550) was measured with a microplate reader. Concentrationswere calculated by comparison with OD550of standard solutions
of sodium nitrite prepared in the cultured medium. 4.4. Cell viability by MTT assay
Cell viability was determined by the mitochondria-dependent reduction of MTT [3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltet-razolium bromide] to formazan. Cells in 96-well plates were incu-bated at 37 °C with MTT (5 mg mL1 for 4 h). Cultured medium
was gently aspirated from each well, and then the MTT crystals were dissolved in acid-SDS (100
l
L). The reduction of the MTT to formazan within the cells were quantitated by measurement of OD570against OD630.4.5. Preparation of human neutrophils
Blood was taken from healthy human donors (20–32 years old) by veinpuncture, using a protocol approved by the institutional re-view board at Chang Gung Memorial Hospital. Neutrophils were isolated with a standard method of dextran sedimentation prior to centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of erythrocytes.22,23Purified neutrophils that contained >98%
via-ble cells, as determined by the trypan blue exclusion method, were re-suspended in a calcium (Ca2+)-free HBSS buffer at pH 7.4, and
were maintained at 4 °C before use.
4.6. Measurement of superoxide anion (O2) generation
The assay of O2generation was based on the SOD-inhibitable
reduction of ferricytochrome c.24In brief, after supplementation with 0.5 mg/ml ferricytochrome c and 1 mM Ca2+, neutrophils were
equil-ibrated at 37 °C for 2 min and incubated with drugs for 5 min. Cells were activated with 100 nM FMLP for 10 min. When FMLP was used as a stimulant, cytochalasin B (CB, 1
l
g/ml) was incubated for 3 min before activation by the peptide (FMLP/CB). Changes in absorbance with the reduction of ferricytochrome c at 550 nm were continu-ously monitored in a double-beam, six-cell positioned spectropho-tometer with constant stirring (Hitachi U-3010, Tokyo, Japan). 4.7. Measurement of elastase releaseDegranulation of azurophilic granules was determined by elastase release as described previously.22 Experiments were performed using MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide as the elastase substrate. Briefly, after supplementation with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100
l
M), neutrophils (6 105ml1) were equilibrated at 37 °C for 2 min and incubated withdrugs for 5 min. Cells were activated by 100 nM FMLP and 0.5
l
g ml1 CB, and changes in absorbance at 405 nm werecontinuously monitored to assay elastase release. The results were expressed as the percentage of elastase release in the FMLP/ CB-activated, drug-free control system.
4.8. Measurement of intracellular calcium concentration ([Ca2+] i)
Neutrophils were loaded with 2
l
M fluo-3 AM at 37 °C for 45 min. After being washed, cells were re-suspended in Ca2+-freeHBSS to 3 106 cells/ml. The change in fluorescence was
moni-tored using a Hitachi F-4500 spectrofluorometer (Tokyo, Japan) in a quartz cuvette with a thermostat (37 °C) and continuous stir-ring. The excitation wavelength was 488 nm, and the emission wavelength was 520 nm. FMLP (0.1
l
M) was used to increase [Ca2+]i in the presence or 1 mM Ca2+. [Ca2+]i was calibrated by
the fluorescence intensity as follows: [Ca2+]
i= Kd [(F Fmin)/
(Fmax F)] where, F is the observed fluorescence intensity, Fmax
and Fmin were obtained by the addition of 0.05% Triton X-100
and 20 mM EGTA, respectively; and Kdwas taken to 400 nM.
Statistical analysis: Results are expressed as the mean ± S.E.M., and comparisons were made using Student’s t-test. A probability of 0.05 or less was considered significant.
Acknowledgments
This work was supported by a grant from National Science Coun-cil, Taiwan, Republic of China, and Grant No.(OUA 95-3-2-021) from the National Cheng Kung University, Tainan, Taiwan R.O.C was awarded to T.S. Wu. This study is supported in part by Taiwan Department of Health Clinical Trail and Research Center of Excellence (DOH100-TD-B-111-004).
Figure 1. Typical traces of the effect of compounds 2 and 21 on mobilization in FMLP-activated human neutrophils. Human neutrophils were incubated with compounds 2 (10lM) and 21 (10lM) for 5 min before stimulation with FMLP. Mobilization of Ca2+
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