Toxicology and Applied Pharmacology 210 (2006) 47 – 54 www.elsevier.com/locate/ytaap

1,2-Naphthoquinone activates vanilloid receptor 1 through increased protein , leading to contraction of guinea pig trachea

Shota Kikunoa,1, Keiko Taguchib,1, Noriko Iwamotob, Shigeru Yamanoc, Arthur K. Chod, John R. Froinesd, Yoshito Kumagaib,d,*

aMaster’s Program in Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan bDepartment of Environmental Medicine, Doctoral Programs in Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan cFaculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan dSouthern California Particle Center and Supersite, Institute of the Environment, University of California, Los Angeles, Los Angeles, CA 90095, USA

Received 7 April 2005; revised 8 June 2005; accepted 10 June 2005 Available online 21 July 2005

Abstract

1,2-Naphthoquinone (1,2-NQ) has recently been identified as an environmental quinone in diesel exhaust particles (DEP) and atmospheric PM2.5. We have found that this quinone is capable of causing a concentration-dependent contraction of tracheal smooth muscle in guinea pigs with EC50 value of 18.7 AM. The contraction required extracellular and was suppressed by L-type calcium channel blockers nifedipine and diltiazem. It was found that 1,2-NQ activated A2 (PLA2)/ (LO)/vanilloid receptor (VR1) signaling. Additionally, 1,2-NQ was capable of transactivating protein tyrosine kinases (PTKs) such as epidermal growth factor receptor (EGFR) in guinea pig trachea, suggesting that phosphorylation of PTKs contributes to 1,2-NQ-induced tracheal contraction. Consistent with this notion, this action was blocked by the PTKs inhibitor genistein and the EGFR antagonist PD153035, indicating that contraction was, at least in part, attributable to PTKs phosphorylation that activates VR1, resulting in increased intracellular calcium content in the smooth muscle cells. D 2005 Elsevier Inc. All rights reserved.

Keywords: 1,2-Naphthoquinone; Diesel exhaust particles; Tracheal contraction; Signal transduction; Vanilloid (capsaicin) receptor 1

Abbreviations: 1,4-BQ, 1,4-benzoquinone; BK, bradykinin; DEP, diesel exhaust particles; EGF, epidermal growth factor; EGFR, epidermal growth Introduction factor receptor; ETYA, 5,8,11,14-eicosatetraynoic acid; FAB-MS, fast atom bombardment mass spectrometry; GSH, reduced glutathione; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; As part of a general study of the toxicological properties LO, lipoxygenase; NMR, nuclear magnetic resonance; NQ, naphthoqui- of reactive substances found in airborne particulate matter none; PAGE, polyacrylamide gel electrophoresis; PBS, -buffered (PM), we have examined selected quinones. Many of the saline; PLA2, phospholipase A2; PM, airborne particulate matter; 9,10-PQ, adverse health effects of PM have been attributed to the 9,10-phenanthraquinone; PTK, protein tyrosine kinase; PTP, protein tyrosine ; pTyr, phosphorylated tyrosine; ROS, reactive oxygen induction of , defined as a change in the species; TBST, Tris-buffered saline containing 0.1% Tween-20; VR1, redox status of the cell (Schafer and Buettner, 2001) vanilloid (capsaicin) receptor 1. commonly assessed by the ratio of oxidized to reduced * Corresponding author. Department of Environmental Medicine, Doc- glutathione and the concentration of reduced glutathione toral Programs in Medical Sciences, Graduate School of Comprehensive (GSH). Quinones can induce oxidative stress in cells by Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Fax: +81 29 853 3133. both changing the ratio through formation of reactive E-mail address: [email protected] (Y. Kumagai). oxygen species (ROS) and by decreasing the GSH concen- 1 Contributed equally to this study. tration through electrophilic reaction (Bolton et al., 2000).

0041-008X/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2005.06.015 48 S. Kikuno et al. / Toxicology and Applied Pharmacology 210 (2006) 47–54

In previous studies (Kumagai et al., 2001), we have (1H at C-7, dt), 7.69 (1H at C-6, dt), 7.87 (1H at C5, dd), found that the redox active quinone, 9,10-phenanthraqui- 8.18 (1H at C-8, dd). FAB-MS spectra were obtained with a none (9,10-PQ), can inhibit nitric oxide synthase in vascular model MS-HX 100 (JEOL Ltd., Tokyo, Japan), and 1H tissue and reduce vascular relaxation. In a study of another NMR-spectra with a model AX-500 (JEOL Ltd.) using PM quinone, 1,2-naphthoquinone (1,2-NQ), we found it to tetramethylsilane as an internal standard at 500 MHz. cause a contraction of tracheal smooth muscle. As is one of the major adverse health effects of PM, this finding Measurement of tracheal contraction. Male Dunkin– was of relevance in the general toxicology of PM. Hartley guinea pigs (250–550 g) were killed by diethyl Furthermore, as the actions of this quinone were persistent, ether inhalation and exsanguination, and each trachea was it was possible that the effects could make pulmonary tissue removed. After removal of and connective tissue, the resistant to dilation, resulting in exacerbation of the asthma trachea was opened longitudinally opposite the trachealis, syndrome. Based on these considerations, we explored the and transverse strips consisting of two adjacent cartilage underlying mechanisms for this effect. In addition to its rings (ca. 3 mm) were prepared. Each ring was mounted in redox properties, 1,2-NQ can also act as an electrophile, a 5 ml organ bath at an initial tension of 1.5 g. The baths forming covalent bonds with thiols, leading to irreversible contained Krebs–Henseleit solution (115 mM NaCl–4.7 inactivation of thiol proteins so we considered the possi- mM KCl–2.5 mM CaCl2 –2.6 mM MgCl2 –1.2 mM bility that the persistent contraction observed with this KH2PO4 –25 mM NaHCO3 –10 mM glucose, pH 7.4) quinone could be due to its electrophilic properties. including 3 AM indomethacin, and the solution was The results indicate that 1,2-NQ, through covalent maintained at 37 -C and gassed with 5% CO2 –95% O2. interaction, caused the phosphorylation of PTKs, thereby Then, tracheal rings were washed with fresh buffer activating PLA2/LO/VR1 signaling and increasing calcium solution every 15 min for a 60 min equilibration period. levels which, in turn, resulted in contraction of tracheal Tension was measured isometrically using a force-displace- smooth muscle. This initial interaction was irreversible so ment transducer (model DSA-603B, Minebea Co. Ltd., that even low levels of this quinone and others with similar Nagano, Japan). The optimal passive load was determined actions could have a cumulative action on tracheal tissue as the contractile response to 40 mM KCl. After the with chronic exposure. tracheal rings showed maximal responses, they were washed by changing the bath fluid 4 times. 1,2-NQ (dissolved in DMSO) was added cumulatively (final Materials and methods concentration of DMSO in the bath was 0.1–3%). Under these conditions, DMSO had no contractile action when Materials. Chemicals were obtained as follows: 1,2-NQ, added alone and did not affect 1,2-NQ-mediated contractile 1,4-NQ from Tokyo Kasei Industries, Ltd. (Tokyo, Japan); action at concentrations of 0.1–3%. Antagonists or 9,10-PQ, 1,4-benzoquinone (1,4-BQ), naphthalene, capsa- inhibitors examined were added 15 min (nifedi- zepine, EGTA, quinacrin, L-703,606, L-659,877, nifedipine, pine, diltiazem), 20 min (genistein, PD153035), 30 min 5,8,11,14-eicosatetraynoic acid (ETYA), indomethacin, dil- (capsazepine, ETYA, quinacrin), or 60 min (L-703,606, L- tiazem, and genistein from Sigma Chemical Co. (St. Louis, 659,877) prior to the addition of 1,2-NQ. MO), anti-EGFR, anti-phosphorylated tyrosine (anti-pTyr) from Zymed Laboratories Inc. (South San Francisco, CA), Phosphorylation of PTKs. After the tracheal rings were BCA Protein assay kit from Pierce Biotechnology Inc. incubated in the Krebs–Henseleit solution including 3 AM (Rockford, IL), and PD153035 from Calbiochem-Novabio- indomethacin in the absence and presence of 1,2-NQ at chem Corp. (San Diego, CA). All other chemicals used were 37 -C under 5% CO2 –95% O2, each ring sample was of the highest grade available. trans-1,2-Dihydroxy-1,2- extensively washed with a lysis buffer (50 mM Tris–HCl dihydronaphtalene was synthesized from 1,2-NQ by the buffer (pH 7.5)–150 mM NaCl–1% Triton X-100–1% method of Platt and Oesch (1983). The 4-mercaptoethanol deoxycholate–0.1% NaN3 –1 mM EGTA–0.4 mM adduct of 1,2-NQ was prepared by mixing it with 2- EDTA–2.5 Ag/ml atropine–2.5 Ag/ml leupeptin–1 mM mercaptoethanol according to the method of Smithgall et al. PMSF–0.2 mM Na3VO4) and then homogenized in the (1988). These synthetic compounds showed >95% purity. lysis buffer. Protein concentration was determined with a The chemical structures of trans-1,2-dihydroxy-1,2-dihy- BCA protein assay kit by the method of Smith et al. dronaphtalene and 1,2-NQ-4-mercaptoethanol adduct char- (1985). The homogenate was centrifuged at 700  g for acterized by FAB-MS and NMR analyses were as follows: 5 min. trans-1,2-dihydroxy-1,2-dihydronaphtalene: m/z 162 (M+); For detection of pTyr in proteins, the 700  g super- 1H-NMR (ppm) 4.48 (1H at C-2, m), 4.82 (1H at C-1, d), natants (0.2 mg/ml) of guinea pig trachea exposed to 5.98 (1H at C-3, dd), 6.45 (1H at C-4, dd), 7.10 (1H at C-5, DMSO, 1,2-NQ, or naphthalene were immunoprecipitated dd), 7.25 (2H at C-6 and C-7, m), 7.58 (1H at C-8, dd); 1,2- using anti-pTyr (4 Ag) and protein A–Sepharose gel (6 mg NQ-4-mercaptoethanol adduct: m/z 234 (M+); 1H-NMR in 30 Al of the lysis buffer) at 4 -C overnight. Then, the (ppm) 3.28 (2H, t), 4.03 (2H, t), 6.50 (1H at C-3, s), 7.59 mixture was centrifuged at 200  g for 1 min, and the gel S. Kikuno et al. / Toxicology and Applied Pharmacology 210 (2006) 47–54 49 was washed with the lysis buffer (0.4 ml  5). The resulting protein A–Sepharose gel was mixed with 2 SDS-PAGE sample buffer (30 Al) and boiled for 5 min. The mixtures were transferred to Ultrafree-MC centrifuged tubes (Milli- pore Corp., Billerica, MA) with a 0.45 Am membrane and then centrifuged. For detection of phosphorylated EGFR, the 700  g supernatants (1 mg/ml) of the guinea pig trachea were incubated with protein A–Sepharose gel (10 mg in 50 Al of the lysis buffer) at 4 -C for 2 h and then centrifuged at 200  g for 1 min to remove proteins that interact with protein A–Sepharose gel. Supernatants (0.9 ml) were incubated with anti-EGFR (4 Ag) at 4 -C overnight and then immunoprecipitated using protein A–Sepharose Fig. 1. Concentration-dependent contraction of guinea pig trachea by 1,2- gel (6 mg in 30 Al of the lysis buffer) at 4 -C for 2 h. This NQ. Tracheal rings from guinea pigs were incubated with increasing protein A–Sepharose gel was processed for Western blot concentration of 1,2-NQ. Results were calculated as the percent of the prior maximum response to KCl (40 mM) and given as mean T SEM (n = 6). analysis as described above. The obtained supernatants were applied to SDS-PAGE (7.5% acrylamide) followed by electrophoresis and Western The alkylating agent N-ethylmaleimide, which reacts blot analysis according to the method of Kyhse-Andersen readily with protein thiol groups, had a contractile action (1984). Bovine serum albumin (1%) and NaN3 (0.05%) in similar to 1,2-NQ, and pretreatment with this agent (50 AM) Tris-buffered saline containing 0.1% Tween-20 (TBST) and resulted in a marked suppression of 1,2-NQ-mediated 3% (w/v) non-fat dry milk in phosphate-buffered saline contraction of guinea pig (90% inhibition, n = 6). (PBS) were used as the blocking solutions for detection of At a concentration of 50 AM, the contraction intensities of pTyr and EGFR, respectively. The blocking was carried out 1,2-NQ, 1,4-NQ, and 1,4-BQ were 75.2 T 0.6%, 63.1 T 7.7%, at 4 -C overnight. Separated proteins were transferred to and 10.6 T 2.2% of maximum response to 40 mM KCl, PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, respectively (n = 4), indicating a structural selectivity of the CA) at 2 mA/cm2 for 60 min and probed with 0.1% anti- effect. The tricyclic quinones, 9,10-PQ and 9,10-anthraqui- pTyr for 2 h, with 0.2% anti-EGFR for 12 h. Detection of none, also present in atmospheric PM2.5 and DEP (Cho et al., antibody to the membrane was with secondary antibodies 2004), and 5,12-naphthacenequinone were without an effect (anti-mouse IgG for pTyr; anti-sheep IgG for EGFR) on tracheal contraction even at 500 AM. Other polycyclic coupled to horseradish peroxidase. Finally, proteins exam- aromatic hydrocarbons such as anthracene, 1,2-benzanthra- ined were detected by using an ECL system (Amersham cene, 2,3-benzofluorene, benzo[a]pyrene, chrysene, diben- Biosciences Corp., Piscataway, NJ), exposing to X-ray films zofuran, 3,6-dimetylphenanthrene, fluoranthrene, 2- (Fuji Photo Film Co. Ltd., Tokyo, Japan). nitropyrene, phenanthrene, and pyrene did not affect tracheal tension under these conditions. Compounds related to 1,2- Statistical analysis. EC50 values were calculated by non- NQ such as 1,2-NQ-4-mercaptoethanol adduct (covalent linear regression using PRISM version 3.0 (GraphPad attachment negative but redox cycling to generate ROS Software Inc., San Diego). Results were expressed as the positive), trans-1,2-dihydroxy-1,2-dihydronaphthalene percent of the maximum response to KCl (40 mM) and (both covalent attachment negative and redox cycling given as mean T SEM. negative), and naphthalene (without quinone moiety) were also without effect on guinea pig trachea at a concentration of 50 AM (data not shown). Taken together, the results suggested Results that the tracheal contraction involved covalent attachment of the quinone group to protein cysteine residues. Contractile action of polycyclic hydrocarbons 1,2-NQ causes activation of VR1 signaling through PLA2/ As shown in Fig. 1, 1,2-NQ caused concentration- LO pathway, thereby increasing intracellular calcium dependent contractions of guinea pig tracheal rings with content an EC50 value of 18.7 AM. The contraction caused by 40 mM KCl was completely abolished by changing the bath Since many organic compounds affect the vanilloid medium, whereas that caused by 1,2-NQ did not return receptor (Szallasi and Blumberg, 1999; Walpole et al., readily to the basal level under the same conditions (data not 1996), its role in the response was examined. As shown in shown). After exposure to 50 AM 1,2-NQ, the contractile Fig. 2A, capsazepine, a competitive antagonist for VR1 response was reduced by repeated washing and became (Bevan et al., 1992; Ellis and Undem, 1994), significantly unstable. This action suggested that the tissue had been suppressed the 1,2-NQ-mediated contraction of guinea pig altered in an irreversible manner. trachea but in a noncompetitive manner. This VR1 antagonist 50 S. Kikuno et al. / Toxicology and Applied Pharmacology 210 (2006) 47–54

receptor antagonists, L-703,606 (NK-1) and L-659,877 (NK-2) (Li and Zhao, 1998), to organ baths containing tracheal rings caused a suppression of 1,2-NQ-mediated contractile action, that is, these antagonists, at 1 AM concentration, reduced the effect of 100 AM 1,2-NQ from 95.7% (control) to 54.9% (data not shown). If tachykinins are released, it is likely that the peptides increase intra- cellular calcium concentration through L-type calcium channels, leading to the contraction of airway smooth muscle (Bayguinov et al., 2003). To confirm that 1,2-NQ action involved L-type calcium channels, the quinone- mediated contraction was examined in calcium-free Krebs–Henseleit solution that contained 3 AM indometha- cin and 0.4 mM EGTA. As shown in Fig. 4A, the contractile action of 1,2-NQ was completely blocked by omission of calcium. Pretreatment with L-type calcium channel antag- onists such as nifedipine and diltiazem (Hirota et al., 2003) also inhibited tracheal contraction caused by 1,2-NQ in a concentration-dependent manner (Figs. 4B, C), thus indicat- ing the involvement of these channels.

Fig. 2. Effects of VR1 antagonist on tracheal contraction of guinea pig by 1,2-NQ. (A) Tracheal rings from guinea pig were incubated in the absence and presence of 10 AM capsazepine at different concentrations of 1,2-NQ. Capsazepine was applied to organ bath for 30 min before the addition of 1,2-NQ. Results were calculated as the percent of the maximum response to KCl (40 mM) and given as mean T SEM (n = 6). (B) Tracheal rings of guinea pig were incubated with 50 AM 1,2-NQ in the absence and presence of capsazepine (10, 100 AM). Capsazepine was applied to organ bath for 30 min before the addition of 1,2-NQ. Results were calculated as the percent of the maximum response to KCl (40 mM) and given as mean T SEM (n = 3). *P < 0.05; **P < 0.01.

(100 AM) completely blocked the contractile action of 50 AM 1,2-NQ (Fig. 2B). There is evidence that vanilloid receptors may be activated by endogenous products of (LOs) such as HPETE, HETE, and B4 (Hwang et al., 2000) and consistent with this notion. ETYA, a specific inhibitor for LO (Craib et al., 2001), reduced the concen- tration-dependent contraction of guinea pig tracheal ring caused by 1,2-NQ (Fig. 3A), also in a noncompetitive manner. The contraction caused by 1,2-NQ (50 AM) was also abolished by the PLA2 inhibitor quinacrin (Alcon et al., 2000) at 100 AM(Fig. 3B). The noncompetitive nature of the Fig. 3. Effects of LO antagonist on tracheal contraction of guinea pig by antagonism suggested that involvement of VR1 and the LO 1,2-NQ. (A) Tracheal rings of guinea pig were incubated with different products in these actions was indirect. concentrations of 1,2-NQ in the presence of 10 AM ETYA. ETYA was It has been reported that activation of VR1 is associated applied to organ bath for 30 min before the addition of 1,2-NQ. Results with release of tachykinins such as which can were calculated as the percent of the maximum response to KCl (40 mM) T induce the contraction of airway smooth muscle (Hwang and given as mean SEM (n = 6). (B) Tracheal rings of guinea pig were incubated with 50 AM 1,2-NQ in the absence and presence of quinacrin (10, and Oh, 2002). We therefore examined the contribution of 100 AM). Quinacrin was applied to organ bath for 30 min before the tachykinin release evoked by VR1 activation to tracheal addition of 1,2-NQ. Results were calculated as the percent of the maximum contraction caused by 1,2-NQ. Addition of tachykinin response to KCl (40 mM) and given as mean T SEM (n = 4). **P < 0.01. S. Kikuno et al. / Toxicology and Applied Pharmacology 210 (2006) 47–54 51

able to transactivate the PTKs that are part of the EGFR system. To evaluate the possibility that 1,2-NQ could cause increased phosphorylated tyrosine in proteins, we examined protein phosphorylation with antibodies against pTyr (Fig. 5A). After exposure of tracheal ring to 1,2-NQ (50 AM) for 5 min, there were several proteins that underwent tyrosine phosphorylation in the tissue. The enhanced phosphoryla- tion of at least four proteins (310 kDa, 240 kDa, 190 kDa, and 140 kDa) in the tracheal ring was observed. Interest- ingly, 1,2-NQ-dependent tracheal contraction was com- pletely blocked by addition of 100 AM genistein, a specific inhibitor of PTKs (Alcon et al., 2000)(Fig. 5B). These results suggest that protein tyrosine phosphorylation is essential for contractile action of 1,2-NQ. We then investigated EGFR transactivation by its increased phos- phorylation in trachea during exposure to 1,2-NQ. As shown in Fig. 6A, 1,2-NQ phosphorylated EGFR at 50 AM, whereas the hydrocarbon naphthalene did not. Con- sistent with this observation, the antagonist of EGFR, PD153035 (Fry et al., 1994), significantly suppressed

Fig. 4. Participation of L-type calcium channel on tracheal contraction of guinea pig by 1,2-NQ. (A) Tracheal rings of guinea pig were incubated with different concentrations of 1,2-NQ in the presence of calcium-free Krebs– Henseleit solution including 3 AM indomethacin and 0.4 mM EGTA. Results were calculated as the percent of the maximum response to KCl (40 mM) and given as mean T SEM (n = 6). (B and C) Tracheal rings of guinea pig were incubated with 50 AM 1,2-NQ in the absence and presence of either nifedipine or diltiazem (1, 50, 100 AM). These compounds were added to the organ bath for 15 min before the addition of 1,2-NQ. Results were calculated as the percent of the maximum response to KCl (40 mM) Fig. 5. Contribution of PTKs phosphorylation to tracheal contraction of T and given as mean SEM (n = 3). *P < 0.05; **P < 0.01. guinea pig by 1,2-NQ. (A) Tracheal rings of guinea pig were incubated with DMSO (lane 1) and 50 AM 1,2-NQ (lane 2) at 37 -C for 5 min. The samples were then homogenized, and the homogenate centrifuged (700 Â g). The 1,2-NQ transactivates PTKs, resulting in tracheal supernatants were then treated with anti-pTyr (4 Ag) and protein A– contraction Sepharose gel (6 mg in 30 Al of the lysis buffer) and allowed to stand at 4 -C overnight. The resulting mixture was treated as described in Materials The activation of protein tyrosine kinases (PTKs) in and methods for Western blot analysis. (B) Tracheal rings of guinea pig the absence of a soluble cognate ligand, referred to as were incubated with 50 AM 1,2-NQ in the absence and presence of either genistein (10, 100 AM). The inhibitor was applied to organ bath for 20 min PTK transactivation, is well documented (Weiss et al., before the addition of 1,2-NQ. Results were calculated as the percent of the 1997). Abdelmohsen et al. (2003) have recently been maximum response to KCl (40 mM) and given as mean T SEM (n = 4). reported that quinones such as 1,4-BQ and menadione are **P < 0.01. 52 S. Kikuno et al. / Toxicology and Applied Pharmacology 210 (2006) 47–54

An immunoprecipitation study with antibodies against EGFR and pTyr revealed that 1,2-NQ did activate EGFR, resulting in tracheal contraction. Furthermore, reduction of the contraction by addition of genistein and PD153035 supported the notion that activation of PTKs, including EGFR, is essential for this response to 1,2-NQ. Bradykinin (BK) is released after tissue injury triggering various defense responses (Couture et al., 2001) and could activate VR1 in bronchial afferents by generation of PLA2/ LO products (Hwang and Oh, 2002). In preliminary studies, we found that the BK receptor antagonist 10 AM bradyzide had a tendency to suppress tracheal contraction of guinea pig caused by 1,2-NQ (S. Kikuno et al., unpublished observation). Thus, it seems likely that 1,2-NQ-mediated activation of PLA2/LO/VR1 signaling, leading to tracheal contraction, may be due to the release of BK triggered by activation of PTKs. Based on these considerations, we speculate that tracheal smooth muscle contraction caused by 1,2-NQ is initiated by activation of PTK, thereby activating Fig. 6. Involvement of EGFR transactivation in contraction of guinea pig trachea by 1,2-NQ. (A) Tracheal rings of guinea pig were incubated with downstream signaling such as PLA2/LO/VR1 followed by DMSO (lane 1), 1,2-NQ (50 AM, lane 2), and naphthalene (50 AM, lane 3) release of tachykinins associated with increased calcium at 37 -C for 5 min. Then, these samples were homogenized, and the 700 Â g level (Fig. 7). supernatants (1 mg/ml) of the guinea pig trachea were incubated with Receptor PTKs signaling (e.g., EGFR) is regulated by A - protein A–Sepharose gel (10 mg in 50 l of the lysis buffer) at 4 C for 2 h protein tyrosine (PTPs) (Ostman and Bohmer, and then centrifuged at 200 Â g for 1 min. Supernatants (0.9 ml) were incubated with anti-EGFR (4 Ag) at 4 -C overnight and then immunopre- 2001; Reynolds et al., 2003). ROS produced in response to cipitated using protein A–Sepharose gel (6 mg in 30 Al of the lysis buffer) activation of either EGFR or insulin receptors has been at 4 -C for 2 h. This protein A–Sepharose gel was processed as described in found to inhibit PTPs and enhance phosphorylation of these Materials and methods for Western blot analysis. (B) Tracheal rings of PTKs (Lee et al., 1998; Bae et al., 1997; Mahadev et al., A guinea pig were incubated with 50 M 1,2-NQ in the absence and presence 2001). Oxidation of the cysteine residue by of either PD153035 (1, 10 AM). The antagonist was applied to organ bath for 20 min before the addition of 1,2-NQ. Results were calculated as the hydrogen peroxide has been identified as a mechanism for percent of the maximum response to KCl (40 mM) and given as mean T negative regulation of PTPs (Lee et al., 1998; Denu and SEM (n = 4). **P < 0.01. Dixon, 1998). A transient oxidation of PTP1B parallels EGFR stimulation (Lee et al., 1998). contraction by 1,2-NQ (50 AM) at a concentration of 10 AM (Fig. 6B).

Discussion

The present study indicates that 1,2-NQ, a component of atmospheric PM2.5, has a novel action in causing the contraction of tracheal smooth muscle from guinea pig. Omission of calcium from an incubation medium drastically suppressed 1,2-NQ-mediated contraction of guinea pig trachea, suggesting the participation of calcium-dependent pathways in the contractile response. Results with L-type calcium channel blockers nifedipine and diltiazem indicated that tracheal contraction caused by 1,2-NQ was, in part, due to increased intracellular calcium concentration in smooth muscle cells. Furthermore, PLA2/LO/VR1 signaling was involved as the contraction was inhibited by pretreatment with capsazepine, ETYA, and quinacrin in a concentration- Fig. 7. A possible mechanism of tracheal contraction through phosphor- dependent manner. ylation of EGFR by 1,2-NQ. BK, bradykinin; EGFR, epidermal growth factor receptor; iCa2+, intracellular calcium; LO, lipoxygenase; 1,2-NQ, We also assessed the PTK activation of EGFR in tracheal 1,2-naphthoquinone; PLA2, phospholipase A2; PTP1B, protein tyrosine preparations, and, as shown in Fig. 5, several proteins in the phosphatase 1B; PTK, protein tyrosine kinase; VR1, vanilloid (capsaicin) trachea were phosphorylated following exposure to 1,2-NQ. receptor 1. S. Kikuno et al. / Toxicology and Applied Pharmacology 210 (2006) 47–54 53

In the present study, we found that 1,2-NQ, 1,4-NQ, and in work environments with high levels of diesel exhaust will 1,4-BQ which form covalent bonds with protein thiols have much higher levels. The exposure to these quinones (Boatman et al., 2000; Zheng et al., 1997; Schmieder et al., can be estimated from the following data: 2003) caused contraction of tracheal smooth muscle of the Human respiratory rate = 720 L/h or 8640 L/day or 8.64 guinea pig, whereas non-alkylating quinones such as 9,10- m3/8 h day. Then, in one such day, exposure to 1,2-NQ PQ that catalyze redox cycling and ROS generation did not. could be as high as 0.1 nmol/day. This exposure would be As mentioned by Abdelmohsen et al. (2003), it is likely that cumulative as the effects are irreversible, and over a quinone-mediated transactivation of PTKs such as EGFR is protracted period of continuous exposure, exposure to high due to disruption of negative regulation of the PTKs. Klotz nanomole quantities will occur, which could then result in and his associates have proposed that transactivation of micromolar concentrations (nmol/ml) in the epithelial cells. EGFR caused by 2-methyl-1,4-NQ (menadione) is attribut- It should also be recognized that people with a predis- able to covalent attachment rather than redox cycling to position to asthmatic attacks may be more sensitive to these yield ROS (Klotz et al., 2002; Abdelmohsen et al., 2003). In inhibitory actions. a cell-free experiment with purified PTP1B, it was shown that 1,2-NQ covalently bound to PTP1B as evaluated by Western blot analysis with anti-1,2-NQ IgG fraction, Acknowledgments resulting in a diminished enzyme activity (N. Iwamoto et al., unpublished observation). Thus, a reasonable explan- This research was supported in part by Grant-in-Aid ation for 1,2-NQ-mediated contraction of guinea pig #15390184 and #15659141 (YK) for scientific research tracheal smooth muscle is that decreased PTP1B activity from the Ministry of Education, Science, Culture and Sports accompanied by covalent binding of 1,2-NQ to reactive of Japan. Although the research described in this article has thiols causes transactivation of PTKs. Indeed, we postulate been funded in part by the United States Environmental that disruption of tracheal tension caused by 1,2-NQ may be Protection Agency through Grant #R827352-01-0 to UCLA, restored by resynthesis of PTP1B. This activation may be it has not been subjected to the Agency’s required peer and associated with PLA2/LO/VR1 signaling that releases policy review and therefore does not necessarily reflect the substance P, resulting in enhanced calcium levels in the views of the Agency, and no official endorsement should be smooth muscle cells. However, it should be noted that inferred. disruption of PTP1B is not the sole factor in quinone- mediated contractile action because 9,10-PQ, which is capable of inhibiting PTP1B activity (Wang et al., 2004) References and phosphorylates EGFR (S. Kikuno et al., unpublished observation), does not have a contractile action of guinea Abdelmohsen, K., Gerber, P.A., von Montfort, C., Sies, H., Klotz, L.O., pig trachea. Thus, we speculate that, unlike 1,2-NQ, 9,10- 2003. Epidermal growth factor receptor is a common mediator of quinone-induced signaling leading to phosphorylation of connexin-43. PQ may affect downstream components (e.g., PLA2, LO, J. Biol. Chem. 278, 38360–38367. VR1, L-type calcium channel, etc.) involved in the signal- Alcon, S., Camello, P.J., Garcia, L.J., Pozo, M.J., 2000. Activation of dependent tracheal contraction, thereby abolishing the tyrosine kinase pathway by vanadate in gallbladder smooth muscle. pharmacological action. Biochem. Pharmacol. 59, 1077–1089. These results demonstrate another process that could Bae, Y.S., Kang, S.W., Seo, M.S., Baines, I.C., Tekle, E., Chock, P.B., Rhee, S.G., 1997. Epidermal growth factor (EGF)-induced generation account for the toxicity of PM and of quinones in general. of hydrogen peroxide. J. Biol. Chem. 272, 217–221. Quinones of certain structures are able to bind to thiol Bayguinov, O., Hagen, B., Sanders, K.M., 2003. Substance P modulates containing proteins and irreversibly inactivate them. In localized calcium transients and membrane current responses in murine contrast to the reversible effects, these actions would be colonic myocytes. Br. J. Pharmacol. 138, 1233–1243. cumulative over time, dependent on protein turnover for Bevan, S., Hothi, S., Hughes, G., James, I.F., Rang, H.P., Shah, K., Walpole, C.S., Yeats, J.C., 1992. Capsazepine: a competitive anta- recovery so that even at very low levels of exposure, gonist of the sensory neurone excitant capsaicin. Br. J. Pharmacol. significant effects could occur over time. As the half life of 107, 544–552. PM in lung tissues is very long (18 days in rats, Sun and Boatman, R.J., English, J.C., Perry, L.G., Fiorica, L.A., 2000. Covalent McClellan, 1984), substantial accumulation could occur in protein adducts of hydroquinone in tissues from rats: identification the course of chronic exposure. and quantitation of sulfhydryl-bound forms. Chem. Res. Toxicol. 13, 853–860. The relationship between concentration and effect Bolton, J.L., Trush, M.A., Penning, T.M., Dryhurst, G., Monks, T.J., 2000. observed in the in vitro studies performed here and actual Role of quinones in toxicology. Chem. Res. Toxicol. 13, 135–160. exposure may be misleading since the actions of 1,2-NQ are Cho, A.K., Di Stefano, E., You, Y., Rodriguez, C.E., Schmitz, D.A., irreversible. Nevertheless, there are data collected from Kumagai, Y., Miguel, A.H., Eiguren-Fernandez, A., Kobayashi, T., several sites in the Los Angeles Basin that may address this Avol, E., Froines, R.J., 2004. Determination of four quinones in diesel exhaust particles, SRM 1649a, and atmospheric PM2.5. Aerosol. Sci. question. In studies to be published, we have found levels of Tech. 38, 1–14. 3 0.02 to 1.7 ng of 1,2-NQ/m in the volatile fraction of Couture, R., Harrisson, M., Vianna, R.M., Cloutier, F., 2001. Kinin ambient air (levels of 1,4-NQ are 2–3 times higher). Levels receptors in pain and . Eur. J. Pharmacol. 429, 161–176. 54 S. Kikuno et al. / Toxicology and Applied Pharmacology 210 (2006) 47–54

Craib, S.J., Ellington, H.C., Pertwee, R.G., Ross, R.A., 2001. A possible Ostman, A., Bohmer, F.D., 2001. Regulation of receptor tyrosine kinase role of lipoxygenase in the activation of vanilloid receptors by signaling by protein tyrosine phosphatases. Trends Cell Biol. 11, in the guinea-pig bronchus. Br. J. Pharmacol. 134, 30–37. 258–266. Denu, J.M., Dixon, J.E., 1998. Protein tyrosine phosphatases: mechanisms Platt, K.L., Oesch, F., 1983. Efficient synthesis of non-K-region trans- of and regulation. Curr. Opin. Chem. Biol. 2, 633–641. dihydrodiols of polycyclic aromatic hydrocarbons from o-quinones and Ellis, J.L., Undem, B.J., 1994. Inhibition by capsazepine of resiniferatoxin- catechols. J. Org. Chem. 48, 265–268. and capsaicin-induced contractions of guinea pig trachea. J. Pharmacol. Reynolds, A.R., Tischer, C., Verveer, P.J., Rocks, O., Bastiaens, P.I., 2003. Exp. Ther. 268, 85–89. EGFR activation coupled to inhibition of tyrosine phosphatases causes Fry, D.W., Kraker, A.J., McMichael, A., Ambroso, L.A., Nelson, J.M., lateral signal propagation. Nat. Cell Biol. 5, 447–453. Leopold, W.R., Connors, R.W., Bridges, A.J., 1994. A specific inhibitor Schafer, F.Q., Buettner, G.R., 2001. Redox environment of the cell as of the epidermal growth factor receptor tyrosine kinase. Science 265, viewed through the redox state of the glutathione /glutathione 1093–1095. couple. Free Radical Biol. Med. 30, 1191–1212. Hirota, K., Hashiba, E., Yoshioka, H., Kabara, S., Matsuki, A., 2003. Schmieder, P.K., Tapper, M.A., Kolanczyk, R.C., Hammermeister, D.E., Effects of three different L-type Ca2+ entry blockers on airway Sheedy, B.R., Denny, J.S., 2003. Discriminating redox cycling and constriction induced by muscarinic receptor stimulation. Br. J. Anaesth. arylation pathways of reactive chemical toxicity in trout hepatocytes. 90, 671–675. Toxicol. Sci. 72, 66–76. Hwang, S.W., Oh, U., 2002. Hot channels in airways: pharmacology of the Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., vanilloid receptor. Curr. Opin. Pharmacol. 2, 235–242. Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, Hwang, S.W., Cho, H., Kwak, J., Lee, S.Y., Kang, C.J., Jung, J., Cho, S., D.C., 1985. Measurement of protein using bicinchoninic acid. Anal. Min, K.H., Suh, Y.G., Kim, D., Oh, U., 2000. Direct activation of Biochem. 150, 76–85. capsaicin receptors by products of lipoxygenases: endogenous capsai- Smithgall, T.E., Harvey, R.G., Penning, T.M., 1988. Spectroscopic cin-like substances. Proc. Natl. Acad. Sci. 97, 155–6160. identification of ortho-quinones as the products of polycyclic aromatic Klotz, L.O., Patak, P., Ale-Agha, N., Buchczyk, D.P., Abdelmohsen, K., trans-dihydrodiol oxidation catalyzed by dihydrodiol dehydrogenase. A Gerber, P.A., von Montfort, C., Sies, H., 2002. 2-Methyl-1,4- potential route of proximate carcinogen . J. Biol. Chem. 263, naphthoquinone, vitamin K(3), decreases gap-junctional intercellular 1814–1820. communication via activation of the epidermal growth factor Sun, J.D., McClellan, R.O., 1984. Respiratory tract clearance of 14C- receptor/extracellular signal-regulated kinase cascade. Cancer Res. labeled diesel exhaust compounds associated with diesel particles or as 62, 4922–4928. a particle-free extract. Fundam. Appl. Toxicol. 4, 388–393. Kumagai, Y., Hayashi, T., Miyauchi, T., Endo, A., Iguchi, A., Kiriya-Sakai, Szallasi, A., Blumberg, P.M., 1999. Vanilloid (capsaicin) receptors and M., Sakai, S., Yuki, K., Kikushima, M., Shimojo, N., 2001. Phenan- mechanisms. Pharmacol. Rev. 51, 159–212. thraquinone inhibits eNOS activity and suppresses vasorelaxation. Am. Walpole, C.S., Bevan, S., Bloomfield, G., Breckenridge, R., James, I.F., J. Physiol.: Regul., Integr. Comp. Physiol. 281, R25–R30. Ritchie, T., Szallasi, A., Winter, J., Wrigglesworth, R., 1996. Kyhse-Andersen, J., 1984. Electroblotting of multiple gels: a simple Similarities and differences in the structure–activity relationships apparatus without buffer tank for rapid transfer of proteins from of capsaicin and resiniferatoxin analogues. J. Med. Chem. 39, polyacrylamide to nitrocellulose. J. Biochem. Biophys. Methods 10, 2939–2952. 203–209. Wang, Q., Dube´, D., Friesen, R.W., LeRiche, T.G., Bateman, K.P., Trimble, Lee, S.R., Kwon, K.S., Kim, S.R., Rhee, S.G., 1998. Reversible inactivation L., Sanghara, J., Pollex, R., Ramachandran, C., Gresser, M.J., Huang, of protein–tyrosine phosphatase 1B in A431 cells stimulated with Z., 2004. Catalytic inactivation of protein tyrosine phosphatase CD45 epidermal growth factor. J. Biol. Chem. 273, 15366–15372. and protein tyrosine phosphatase 1B polyaromatic quinones. Biochem- Li, H.S., Zhao, Z.Q., 1998. Small sensory neurons in the rat dorsal root istry 43, 4294–4303. ganglia express functional NK-1 tachykinin receptor. Eur. J. Neurosci. Weiss, F.U., Daub, H., Ullrich, A., 1997. Novel mechanisms of RTK signal 10, 1292–1299. generation. Curr. Opin. Genet. Dev. 7, 80–86. Mahadev, K., Wu, X., Zilbering, A., Zhu, L., Lawrence, J.T.R., 2001. Zheng, J., Cho, M., Jones, A.D., Hammock, B.D., 1997. Evidence of Hydrogen peroxide generated during cellular insulin stimulation is quinone metabolites of naphthalene covalently bound to sulfur integral to activation of the distal insulin signaling cascade in 3T3-L1 nucleophiles of proteins of murine clara cells after exposure to adipocytes. J. Biol. Chem. 276, 48662–48669. naphthalene. Chem. Res. Toxicol. 10, 1008–1014.