* Institute of Toxicology and
Department of Orthopaedics, College of Medicine, National Taiwan University, Taipei, Taiwan
Received April 10, 2003; accepted May 28, 2003
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ABSTRACT |
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Key Words: motorcycle exhaust particulate extract; aorta; vascular smooth muscle cell; myosin light chain kinase; reactive oxygen species.
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INTRODUCTION |
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The major regulatory mechanism of smooth muscle contraction and relaxation is phosphorylation and dephosphorylation of the 20-kDa myosin light chain (MLC20) (Allen et al., 1994). MLC20 is phosphorylated by the Ca2+-calmodulin (CaM)activated myosin light chain kinase (MLCK) and dephosphorylated by the Ca2+-independent myosin light chain phosphatase (MLCP). Thus, a rise in cytosolic Ca2+ concentration ([Ca2+]i) produces smooth muscle contraction by binding to calmodulin, the activation of MLCK, and the subsequent phosphorylation of MLC20 (Fukata et al., 2001
; Wilson et al., 2002
). The Ca2+ influx plays a major role in maintaining the sustained [Ca2+]i elevation during contractions. The L-type Ca2+ channel is considered to be a major Ca2+ influx pathway and is responsible for vascular smooth muscle contractility (Karaki et al., 1997
; Triggle et al., 1998
). Furthermore, reactive oxygen species (ROS) may also participate in the regulation of vascular tone (Suzuki and Ford, 1999
). It has been indicated that ROS generated by different compounds or systems could induce vasoconstriction in various arteries such as rat aortas (Peters et al., 2000
; Shen et al., 2000
), porcine coronary arteries (Grover et al., 1999
), rabbit carotid arteries (Heinle, 1984
), rat and porcine pulmonary arteries (Jin et al., 1997
; Sanderud et al., 1991
), canine basilar arteries (Tosaka et al., 2002), and human umbilical arteries (Okatani et al., 1997). These studies provide evidence that H2O2 or other ROS may be the vasoactive mediators in arteries by acting as second messengers. A recent report further suggested that ROS generated by mitochondria appear to function as second messengers during hypoxia and act to trigger the Ca2+-signaling process responsible for the contraction of pulmonary arterial myocytes during hypoxia (Waypa et al., 2002
). Therefore, the present study was performed to investigate the effect of MEPE on the contractile response in isolated rat aortas, to examine whether the Ca2+MLCK pathway is involved, and to determine the role of ROS in the MEPE-induced response.
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MATERIALS AND METHODS |
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Aorta culture.
Male Wistar rats (200250 g) were purchased from the Animal Center of the College of Medicine, National Taiwan University, Taipei, Taiwan. The Animal Research Committee of National Taiwan University, College of Medicine, conducted the study in accordance with the guidelines for the care and use of laboratory animals. The rats were anesthetized with sodium pentobarbital, and the thoracic aorta was removed, cleaned of fat and adventitia, and cut into ring segments of 45 mm length with parallel razors. When necessary, the endothelium was carefully removed by gentle rubbing of the luminal surface with a cotton-tipped applicator. These isolated aortic rings were cultured in an organ culture petri dish (Falcon) with sterile Dulbeccos modified Eagles medium (DMEM), containing 10% fetal bovine serum and 1% antibiotic solution at 37°C. After 18 h, the rings were prepared for organ bath study. MEPE were dissolved in dimethylsulfonyl oxide (DMSO) and added to the medium. The concentration of DMSO in the medium was less than 0.1%.
Organ bath study.
The vasoconstriction of aorta rings was measured by the method described by Liu et al. (1999). The aorta rings were suspended between two hooks connected to a transducer (Grass FT.03) for the measurement of isometric force. The rings were suspended in 10-ml organ baths containing oxygenated (95% O2 + 5% CO2) and warmed (37°C) Krebs solution containing (composition in mM) NaCl 118.3, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25.0, and glucose 11.1. The pH of the Krebs solution was 7.27.4. The basal tension was set at 1.0 g. The rings were allowed to equilibrate for 1 h before a concentrationresponse curve to phenylephrine (0.00310 µM) was obtained. The tension was recorded by an isometric transducer (Grass FT.03) on a Biopacs MP 100 data acquisition system with analytic software (AcqKnowledge, Version 3.0, Biopac Systems Inc., Ste E Goleta, CA), the outputs of which were written on an HP deskjet 500C printer. In some experiments, inhibitors were added simultaneously with the MEPE treatment. The absence of endothelium was confirmed by lack of a response to acetylcholine.
Primary vascular smooth muscle cell culture.
Vascular smooth muscle cells (VSMCs) were obtained from the thoracic aortas of Wistar rats by the method described by Bierman et al. (1974). In brief, male rats (200250 g) were sacrificed and the thoracic aortas were removed, cleaned of fat and adventitia, cut into small strips, and then incubated in 1 mg/ml collagenase and 0.125 mg/ml elastase at 37°C for 60 min. The cells were seeded into 10-mm diameter dishes and maintained in 10 ml of DMEM containing 10% fetal bovine serum at 37°C. The cells were used between the third and sixth passages. The cells exhibited characteristics of VSMCs. The cells were grown to 60 to 80% confluence, at which time they were rendered quiescent by the DMEM medium containing 0.1% bovine serum albumin (BSA) and maintained for 48 h before experimentation.
Measurement of MLC20 phosphorylation.
The extent of MLC phosphorylation in VSMCs was determined using the urea-glycerol gel electrophoresis technique (Persechini et al., 1986), followed by immunoblot detection with a specific mouse monoclonal anti-MLC antibody (Sigma). In brief, the VSMCs were transferred to a denaturation solution consisting of 10% trichloroacetic acid in acetone and 10-mM dithiothreitol pre-chilled at -80°C. The VSMCs were then washed three times extensively and stored in acetone containing 10-mM dithiothreitol at -80°C. After the VSMCs were dried to remove the acetone, they were extracted in the sample buffer (8-M urea, Tris [hydroxymethyl] aminomethare 20 mM, glycine 23 mM, 0.004% bromophenol blue and dithiothreitol 10 mM) at room temperature for 2 h. The supernatant was subjected to electrophoresis on 10% polyacrylamide gel containing 40% glycerol, followed by transfer onto a nitrocellulose membrane. The 20-kDa MLC, both unphosphorylated and phosphorylated, was detected by a specific antibody (1:3000) and a horse radish peroxidase-conjugated secondary antibody (1:7500). An immune complex was detected by using an enhanced chemiluminescence technique (Amersham, Buckinghamshire, UK).
Detection of intracellular ROS.
Intracellular ROS generation was monitored by flow cytometry using a peroxide-sensitive fluorescent probe (2',7'-dichlorofluorescin diacetate (DCFH-DA), Molecular Probes, Eugene, OR) as described in Lund-Johansen and Oluweus (1992). In brief, the experiments were performed under dim light. Subconfluent and serum-deprived VSMCs were loaded with 10 µM of DCFH-DA for 30 min after the treatment of MEPE and then chilled on ice and washed with cold PBS. The washed cells were detached from the culture plates by trypsin digestion. The fluorescent intensities for samples of 10,000 cells each was analyzed by flow cytometry with the use of a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.
Statistics.
The values given are as mean ± SEM. The significance of difference from the respective controls for each experimental test condition was assessed by using one-way analysis of variance followed by Dunnetts test for each paired experiment. Patterns of vasoconstriction were analyzed by two-way ANOVA with repeated measurements. P values < 0.05 were regarded as indicating significant differences.
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RESULTS |
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DISCUSSION |
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The primary signal for smooth muscle contraction is an increase in sarcoplasmic free Ca2+ concentration [Ca2+]i. This triggers activation of Ca2+-dependent myosin light chain kinase. In smooth muscle, an L-type Ca2+ channel is considered to be a major Ca2+ influx pathway (Karaki et al., 1997). The L-type Ca2+ channel is responsible for normal myocardial contractility and vascular smooth muscle contractility (Triggle 1998
). In the present study, we found that manganese and L-type Ca2+ channel blocker nifidepine significantly suppressed MEPE-enhanced vasoconstriction in organ cultures of rat aortas. Therefore, the mechanism of MEPE-induced enhancement of vasoconstriction may be related to the alteration in the transmembranous influx of Ca2+. However, although inhibiting a physiological mediator may block the response, this alone does not prove that the test agent is operating through that particulate physiological mediator; it just proves that the mediator is required for the response. Therefore, the direct correlation between the increase of Ca2+ influx and vasoconstriction in the presence of MEPE needs further investigation. An increase in [Ca2+]i saturates the four Ca2+-binding sites of CaM, which then binds to and activates actin-bound MLCK. The activated the MLCK phosphorylates myosin light chain, and then phosphorylated myosin interacted with actin to induce the contractile response (Karaki et al., 1997
; Wilson et al., 2002
). Moreover, it has also been suggested that PKC could increase Ca2+ sensitivity through the inhibition of MLC phosphatase in smooth muscles (Karaki et al., 1997
; Morgan and Leinweber, 1998
). Our present study showed that staurosporine, a nonselective PKC inhibitor, could inhibit the MEPE-enhanced vasoconstriction; however, chelerythrine, a potent and selective PKC inhibitor (Herbert et al., 1990
), failed to abolish the stimulatory effect of MEPE. These results imply that a PKC pathway is not involved in the enhancement of vasoconstriction by MEPE. It has been reported that staurosporine could also act as an MLCK inhibitor at IC50 1.3 nM (Tamaoki et al., 1986
). Our further results showed that both nonselective MLCK inhibitor staurosporine and selective MLCK inhibitor ML-9 suppress the MEPE-enhanced constriction in rat aortas. We also identified that MEPE was capable of inducing the phosphorylation of MLC20 in cultured vascular smooth muscle cells, which could be blocked in the presence of ML-9. Therefore, these findings indicate that a Ca2+/CaM/MLCK-dependent pathway is involved in the MEPE-enhanced vasoconstriction in aortas.
Increasing evidence suggests that reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide, and hydroxyl radical, are produced by a variety of cell types and may modulate physiological and pathophysiological processes (Schnackenberg, 2002). Recent studies have shown that ROS may function as a second messenger in the activation of transcriptional factors, gene expression, cell growth, chemotaxis, and apoptosis (Irani, 2000
). These ROS also have direct vasocontractile effects on several vessels, such as rat aortas (Peters et al., 2000
; Shen et al., 2000
; Yang et al., 1998
). Some studies have identified that H2O2 was capable of evoking vasoconstriction through a Ca2+-dependent mechanism in rat aortas (Sotnikova, 1998
; Yang et al., 1998
). A recent report further showed that catalase prevents elevation of [Ca2+]i induced by alcohol in cultured canine cerebral vascular smooth muscle cells, which may indicate a possible relationship to alcohol-induced strokes and brain pathology (Li et al., 2003
). Waypa et al. (2002)
have also suggested that ROS generated by mitochondria appear to function as second messengers during hypoxia and act to trigger the Ca2+-signaling process responsible for the contraction of pulmonary arterial myocytes during hypoxia. It has been demonstrated that DEPs might stimulate ROS generation in macrophage-like cell lines RAW 264.7 and bronchial epithelial cells (Hiura et al., 1999
; Li et al., 2002
). Indeed, MEPE have also been reported to stimulate ROS generation in various cell systems, including hepatic, pulmonary, and macrophage-like cell lines (Kuo et al., 1998
; Lee et al., 2002
). In the present study, we found that antioxidant NAC significantly inhibited MEPE-enhanced vasoconstriction in rat aortas, and MEPE could stimulate ROS generation in a time-dependent manner in cultured vascular smooth muscle cells. Therefore, these findings suggest that ROS induced by MEPE may contribute to Ca2+ influx and MLCK activation and subsequently enhance vasoconstriction.
There are more than 100 C1C16 hydrocarbons including PAHs in motorcycle emissions (Jemma et al., 1995; Ueng et al., 2000
). It has been reported that short-term treatment of some PAHs could induce vasorelaxation of rat aortas in an endothelium-dependent manner (Kang and Cheng, 1997
). Therefore, there is the need for more work on the chemical characterization of an MEPE sample followed by subsequent bioactivity testing of the primary chemical constituent groups in the future.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Bai, Y., Suzuki, A. K., and Sagai, M. (2001). The cytotoxic effects of diesel exhaust particles on human pulmonary artery endothelial cells in vitro: Role of active oxygen species. Free Radic. Biol. Med. 30, 555562.[CrossRef][ISI][Medline]
Bierman, E. L., Stein, O., and Stein, Y. (1974). Lipoprotein uptake and metabolism by rat aortic smooth muscle cells in tissue culture. Circ. Res. 35, 136150.[ISI][Medline]
Casellas, M., Fernandez, P., Bayona, J. M., and Solanas, A. M. (1995). Bioassay-directed chemical analysis of genotoxic components in urban airborne particulate matter from Barcelona (Spain). Chemosphere 30, 725740.[CrossRef][ISI][Medline]
Cheng, Y. W., and Kang, J. J. (1999). Inhibition of agonist-induced vasoconstriction and impairment of endothelium-dependent vasorelaxation by extract of motorcycle exhaust particles in vitro. J. Toxicol. Environ. Health A 57, 7587.[CrossRef][ISI][Medline]
Environment Protection Agency, R.O.C. (2000). Environmental Data of Taiwan in 1994, R.O.C., pp. 3177. Environmental Protection Agency, Taipei.
Escobal, A., Iriondo, C., and Laborra, C. (1997). Determination of volatile compounds in Txakoli wine from Biscay by gas chromatography-mass spectrometry. J. Chromatogr. A 778, 225234.[CrossRef][ISI][Medline]
Fukata, Y., Amano, M., and Kaibuchi, K. (2001). Rho-Rho-kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non-muscle cells. Trends Pharmacol. Sci. 22, 3239.[CrossRef][ISI][Medline]
Grover, A. K., Samson, S. E., Misquitta, C. M., and Elmoselhi, A. B. (1999). Effects of peroxide on contractility of coronary artery rings of different sizes. Mol. Cell Biochem. 194, 159164.[CrossRef][ISI][Medline]
Heinle, H. (1984). Vasoconstriction of carotid artery induced by hydroperoxides. Arch. Int. Physiol Biochim. 92, 267271.[ISI][Medline]
Herbert, J. M., Augereau, J. M., Gleye, J., and Maffrand, J. P. (1990). Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem. Biophys. Res. Commun. 172, 993999.[ISI][Medline]
Hiura, T. S., Kaszubowski, M. P., Li, N., and Nel, A. E. (1999). Chemicals in diesel exhaust particles generate reactive oxygen radicals and induce apoptosis in macrophages. J. Immunol. 163, 55825591.
Hricko, A. (1994). Toxic Taipei traffic. Environ. Health Perspect. 102, 279.
Ikeda, M., Suzuki, M., Watarai, K., Sagai, M., and Tomita, T. (1995). Impairment of endothelium-dependent relaxation by diesel exhaust particles in rat thoracic aorta. Jpn. J. Pharmacol. 68, 183189.[ISI][Medline]
Irani, K. (2000). Oxidant signaling in vascular cell growth, death, and survival: a review of the roles of reactive oxygen species in smooth muscle and endothelial cell mitogenic and apoptotic signaling. Circ. Res. 87, 179183.
Jemma, C. A., Shore, P. R., and Widdicombe, K. A. (1995). Analysis of C1C16 hydrocarbons using dual-column capillary G.C.: Application to exhaust emissions from passenger car and motorcycle engine. J. Chromatogr. Sci. 33, 3448.[ISI]
Jeng, Y.-J., and Peng, F.-J. (1997). Chemicla analysis and identification of motorcycle exhaust particulates. 12th Joint Annual Conference on Biomedical Science, Taipei, Taiwan, 234 (abstract).
Jin, N., and Rhoades, R. A. (1997). Activation of tyrosine kinases in H2O2-induced contraction in pulmonary artery. Am. J. Physiol. 272, H2686H2692.[ISI][Medline]
Kang, J. J., and Cheng, Y. W. (1997). Polycyclic aromatic hydrocarbons-induced vasorelaxation through activation of nitric oxide synthase in endothelium of rat aorta. Toxicol. Lett. 93, 3945.[CrossRef][ISI][Medline]
Karaki, H., Ozaki, H., Hori, M., Mitsui-Saito, M., Amano, K., Harada, K., Miyamoto, S., Nakazawa, H., Won, K. J., and Sato, K. (1997). Calcium movements, distribution, and functions in smooth muscle. Pharmacol. Rev. 49, 157230.
Kuo, M. L., Jee, S. H., Chou, M. H., and Ueng, T. H. (1998). Involvement of oxidative stress in motorcycle exhaust particle-induced DNA damage and inhibition of intercellular communication. Mutat. Res. 413, 143150.[ISI][Medline]
Lee, C. C., and Kang, J. J. (2002). Extract of motorcycle exhaust particles induced macrophages apoptosis by calcium-dependent manner. Chem. Res. Toxicol. 15, 15341542.[CrossRef][ISI][Medline]
Li, H., Banner, C. D., Mason, G. G., Westerholm, R. N., and Rafter, J. J. (1996). Determination of polycyclic aromatic compounds and dioxin receptor ligands present in diesel exhaust particulate extracts. Atmos. Environ. 30, 35373543.[CrossRef][ISI]
Li, N., Wang, M., Oberley, T. D., Sempf, J. M., and Nel, A. E. (2002). Comparison of the pro-oxidative and proinflammatory effects of organic diesel exhaust particle chemicals in bronchial epithelial cells and macrophages. J. Immunol. 169, 45314541.
Li, W., Liu, W., Altura, B. T., and Altura, B. M. (2003) Catalase prevents elevation of [Ca2+]i induced by alcohol in cultured canine cerebral vascular smooth muscle cells: Possible relationship to alcohol-induced stroke and brain pathology. Brain. Res. Bull. 59, 315318.[CrossRef][ISI][Medline]
Liu, S. H., Tzeng, H. P., Kuo, M. L., and Lin-Shiau, S. Y. (1999). Inhibition of inducible nitric oxide synthase by beta-lapachone in rat alveolar macrophages and aorta. Br. J. Pharmacol. 126, 746750.
Liu, S. H., Wang, J. H., Chuu, J. J., and Lin-Shiau, S. Y. (2002). Alterations of motor nerve functions in animals exposed to motorcycle exhaust. J. Toxicol. Environ. Health A 65, 803812.[CrossRef][ISI][Medline]
Lund-Johansen, F., and Oluweus, J. (1992). Signal transduction in monocytes and granulocytes measured by multiparameter flow cytometry. Cytometry 13, 693702.[ISI][Medline]
Morgan, K. G., and Leinweber, B. D. (1998). PKC-dependent signalling mechanisms in differentiated smooth muscle. Acta Physiol Scand. 164, 495505.[CrossRef][ISI][Medline]
Nel, A. E., Diaz-Sanchez, D., Ng, D., Hiura, T., and Saxon, A. (1998). Enhancement of allergic inflammation by the interaction between diesel exhaust particles and the immune system. J. Allergy Clin. Immunol. 102, 539554.[ISI][Medline]
Persechini, A., Kamm, K. E., and Stull, J. T. (1986). Different phosphorylated forms of myosin in contracting tracheal smooth muscle. J. Biol. Chem. 261, 62936299.
Peters, S. L., Mathy, M. J., Pfaffendorf, M., and van Zwieten, P. A. (2000). Reactive oxygen species-induced aortic vasoconstriction and deterioration of functional integrity. Naunyn Schmiedebergs Arch. Pharmacol. 361, 127133.[CrossRef][ISI][Medline]
Sanderud, J., Norstein, J., and Saugstad, O. D. (1991). Reactive oxygen metabolites produce pulmonary vasoconstriction in young pigs. Pediatr. Res. 29, 543547.[Abstract]
Schnackenberg, C. G. (2002). Physiological and pathophysiological roles of oxygen radicals in the renal microvasculature. Am. J. Physiol Regul. Integr. Comp. Physiol. 282, R335R342.
Shen, J. Z., Zheng, X. F., and Kwan, C. Y. (2000). Differential contractile actions of reactive oxygen species on rat aorta: Selective activation of ATP receptor by H2O2. Life Sci. 66, L291L296.[CrossRef]
Sotnikova, R. (1998). Investigation of the mechanisms underlying H2O2-evoked contraction in the isolated rat aorta. Gen. Pharmacol. 31, 115119.[CrossRef][ISI][Medline]
Suzuki, Y. J., and Ford, G. D. (1999). Redox regulation of signal transduction in cardiac and smooth muscle. J. Mol. Cell Cardiol. 31, 345353.[CrossRef][ISI][Medline]
Tamaoki, T., Nomoto, H., Takahashi, I., Kato, Y., Morimoto, M., and Tomita, F. (1986). Staurosporine, a potent inhibitor of phospholipid/Ca2+ dependent protein kinase. Biochem. Biophys. Res. Commun. 135, 397402.[ISI][Medline]
Terada, N., Hamano, N., Maesako, K. I., Hiruma, K., Hohki, G., Suzuki, K., Ishikawa, K., and Konno, A. (1999). Diesel exhaust particulates upregulate histamine receptor mRNA and increase histamine-induced IL-8 and GM-CSF production in nasal epithelial cells and endothelial cells. Clin. Exp. Allergy 29, 5259.[CrossRef][ISI][Medline]
Triggle, D. J. (1998). The physiological and pharmacological significance of cardiovascular T-type, voltage-gated calcium channels. Am. J. Hypertens. 11, 80S87S.[CrossRef][Medline]
Ueng, T. H., Hu, S. H., Chen, R. M., Wang, H. W., and Kuo, M. L. (2000). Induction of cytochrome P-450 1A1 in human hepatoma HepG2 and lung carcinoma NCI-H322 cells by motorcycle exhaust particulate. J. Toxicol. Environ. Health A 60, 101119.[CrossRef][ISI][Medline]
Ueng, T. H., Hwang, W. P., Chen, R. M., Wang, H. W., Kuo, M. L., Park, S. S., and Guengerich, F. P. (1998). Effects of motorcycle exhaust on cytochrome P-450-dependent monooxygenases and glutathione S-transferase in rat tissues. J. Toxicol. Environ. Health A 54, 509527.[CrossRef][Medline]
Waypa, G. B., Marks, J. D., Mack, M. M., Boriboun, C., Mungai, P. T., and Schumacker, P. T. (2002). Mitochondrial reactive oxygen species trigger calcium increases during hypoxia in pulmonary arterial myocytes. Circ. Res. 91, 719726.
Wilson, D. P., Sutherland, C., and Walsh, M. P. (2002). Ca2+ activation of smooth muscle contraction: Evidence for the involvement of calmodulin that is bound to the triton insoluble fraction even in the absence of Ca2+. J. Biol. Chem. 277, 21862192.
Yang, Z. W., Zheng, T., Zhang, A., Altura, B. T., and Altura, B. M. (1998). Mechanisms of hydrogen peroxide-induced contraction of rat aorta. European J. Pharmacol. 344, 169181.[CrossRef][ISI][Medline]
Zhou, W., and Ye, S. H. (1997). Mutagenicity of scooter exhaust particulate matter. J. Toxicol. Environ. Health A 52, 3544.[CrossRef]