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Roles of Inhibitors of Myosin Light Chain Kinase and Tyrosine Kinase on Cation Influx in Agonist-Stimulated Endothelial Cells
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
235, 657–662 (1997)
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Roles of Inhibitors of Myosin Light Chain Kinase and Tyrosine Kinase on Cation Influx in Agonist-Stimulated Endothelial Cells Reiko Takahashi,1 Hiroshi Watanabe, Xu-Xia Zhang, Hiroyasu Kakizawa, Hideharu Hayashi, and Ryuzo Ohno Internal Medicine III, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-31, Japan
Received May 6, 1997
Agoinst-stimulated Ca2/ influx is critically important to mediate the function of endothelial cells. It has been suggested that release of Ca2/ from internal stores activates Ca2/ influx across the plasma membrane. In the present study, we investigated the effects of ML-9, a myosin light-chain kinase (MLCK) inhibitor, and genistein, a tyrosine kinase inhibitor, on the agonist stimulated Ca2/ response in porcine aortic endothelial cells loaded with a Ca2/-sensitive dye, fura-2. ML-9 almost completely abolished Ca2/ influx, whereas genistein only partially attenuated Ca2/ entry. Both of them did not affect the mobilization of Ca2/ from internal stores. In contrast, genistein was more potent in the inhibition of Mn2/ influx than ML-9. These findings indicate the different selectivity for Ca2/ and Mn2/ in the cation entry pathway in agonist-stimulated endothelial cells. q 1997 Academic Press
Endothelial cells are multifunctional cells that regulate permeability, between the plasma and the interstitial space, vascular tone, by releasing endothelial derived relaxing and/or constricting factors, and are involved in angiogenesis, anticoagulation and inflammatory responses (1-3). The cytosolic calcium ion is one of the major second messengers that mediate the functions of endothelial cells. The intracellular concentration of Ca2/([Ca2/]i) shows biphasic increases in response to agonist stimulation with a rapid initial increase followed by a sustained increase. The initial increase [Ca2/]i is largely dependent on the release of calcium from Inositol 1,4,5-Triphosphate (Ins(1,4,5)P3)sensitive stores (4,5). The sustained increase in [Ca2/]i 1 To whom correspondence should be addressed. Fax: /81 53 434 2910. E-mail: [email protected]. Abbreviations used: MLC, myosin light-chain; MLCK, myosin light-chain kinase; Ins(1,4,5)P3 , inositol 1,4,5-triphosphate.
is caused by Ca2/ influx from the extracellular space. Although it has been suggested that the influx of Ca2/ can be triggered by the depletion of internal Ca2/ stores (6-8), the mechanism(s) communicating the release of Ca2/ from intracellular stores with the influx of Ca2/ from extracellular space is not well understood. Recently we reported that myosin light-chain kinase (MLCK) inhibitors, ML-9 and wortmannin inhibited Ca2/ influx from the extracellular space without affecting the release of Ca2/ from internal stores in bradykinin- and thapsigargin-treated porcine aortic endothelial cells (9). The data indicate that a common part of the signal transduction cascade initiated by a receptordependent and -independent stimulation of endothelial cells includes a MLCK inhibitor-sensitive influx of Ca2/ across the plasma membrane. On the other hand, it has been shown that inhibition of protein tyrosine phosphorylation by different tyrosine kinase inhibitors, herbimycin A, genistein and piceatannol attenuated agonist-stimulated Ca2/ entry in venous endothelial cells from human umbilical cords (10,11). However, the potency these inhibitors to inhibit the Ca2/ influx was not clarified in agonist-stimulated endothelial cells (12,13). Therefore, one of the purposes of this study is to determine which kinase inhibitor plays the major role in the regulation of the influx of Ca2/ in endothelial cells. Agonist stimulation has been shown to enhance not only Ca2/ influx but also Mn2/ influx into the cells in the presence of extracellular Mn2/. Mn2/ influx is assessed by the quenching of the fluorescence of fura-2 (14-16). The pathway for this cation entry remains to be determined. In our previous study, the influx of Ca2/ caused by a receptor-independent agonist thapsigargin was attenuated by either membrane depolarization due to a high K/ solution or the pretreatment of endothelial cells with NiCl2 , but was not influenced by verapamil a blocking agent of voltage dependent Ca2/ channel nor the replacement of Na/ by Li/ in the extracellular
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solution (17). These results suggest the involvement of non-specific cation channels independent of the voltage-dependent Ca2/ channels and/or Na//Ca2/ exchange mechanisms (18,19). Another purpose of this study is to evaluate whether inhibitors of MLCK and tyrosine kinase can modify the influx of other cations such as Mn2/ across the plasma membrane. We report in the present study that the MLCK inhibitor, ML-9 almost completely inhibits the Ca2/ influx including tyrosine kinase inhibitor-insensitive component in bradykinin-stimulated endothelial cells, suggesting that MLCK is potentially more important than tyrosine kinase in the regulation of Ca2/ influx in endothelial cells. In contrast, tyrosine kinase inhibitor, genistein is more potent in the inhibition of Mn2/ influx than the MLCK inhibitor. These findings indicate the different selectivity for Ca2/ and Mn2/ in the cation entry pathway in agonist-stimulated endothelial cells. MATERIALS AND METHODS Cell culture. Porcine aortic endothelial cells were isolated, as previously described (20,21) by gentle mechanical scraping the intima of the descending part of porcine aortas. After centrifugation at 250 1 g for 10 min in M199 solution, the fraction of endothelial cells was purified from this suspension, resuspended in M199 solution with Earle’s salts, supplemented 100 IU/ml penicillin G, 100 mg/ml streptomycin, and 20 % newborn calf serum (NCS), then aliquotted into the polybiphenyl dishes fixed on 10 1 10-mm glass cover slips, and cultured in an incubator at 377C under 5% CO2 for two days. The medium was renewed every day. Measurement of cytosolic calcium concentration. [Ca2/]i was measured, as previously described (17) in endothelial cells adhering to the glass cover slip. The cells were incubated for 45 minutes in modified Tyrode’s solution (composition in mM: 150.0 NaCl, 2.7 KCl, 1.2 KH2PO4 , 1.2 MgSO4 , 1.0 CaCl2 , and 10.0 N-2-hydroxyethyl-piperazine-N*-2-ethanesulfonic acid, with pH 7.4 at 257C) containing 10% NCS and 2mM fura-2/AM, a fluorescent Ca2/ indicator. The cells were subsequently washed three times with modified Tyrode’s solution to remove the fura-2/AM and the serum from the extracellular fluid followed by a 20-min postincubation period before measurements were started. All experiments were performed at 257C. The absorption shift of fura-2 that occurs upon binding can be determined by scanning the excitation spectrum between 340 and 380 nm while monitoring the emission at 510 nm. The fluorescent image was analyzed every 30 s from the individual cells with an [Ca2/]i analyzer (Argus 50, Hamamatsu Photonics) using a ultra-high sensitivity television camera (CCD). The fluorescence ratio (F340/ F380) was obtained by dividing, pixel by pixel, the 340-nm image after background subtraction by the 380-nm image after background subtraction. Intracellular calibration was performed in accordance with the method of Li et al. (22). In order to obtain the maximum Rmax or minimum Rmin value of the fluorescence ratio, after fura-2 loading, the endothelial cells were exposed to modified Tyrode’s solution containing 10 mM ionomycin and 3 mM of Ca2/ or 5 mM ethylene glycol-bis(ßaminoethyl ether)-N,N,N*,N*-tetraacetic acid (EGTA), respectively. [Ca2/]i was calculated from the equation of Grynkiewicz et al. (23). Bradykinin, thapsigargin, ML-9, and genistein had no effect on fura2 fluorescence itself, or on autofluorescence of unloaded cells when examined at concentrations employed in this study. Measurement of influx of Mn2/. Fura-2 has a higher affinity for Mn2/ (Kd Å 5.33 nM) than for Ca2/ (23). If Mn2/ enters into cells, the binding of Mn2/ with fura-2 quenches its fluorescence. Since the
absorption of fura-2 excited at 360 nm is not affected depending on [Ca2/]i , the decline of fluorescence excited at 360 nm can be utilized to monitor Mn2/ influx into cells (17). Materials. Medium 199, newborn calf serum and penicillin-streptomycin were purchased from GIBCO (New York, USA), fura-2/AM was from Dojindo (Kumamoto, Japan), and from Wako(Osaka, Japan). Bradykinin, thapsigargin, ML-9, and genistein were from Sigma (St. Louis, USA). All other chemicals were of analytical grade.
RESULTS Effect of genistein and ML-9 on bradykinin-stimulated Ca2/ response. The effect of the tyrosine kinase inhibitor genistein on bradykinin-stimulated Ca2/ response was contrasted with the effect of the myosin light chain kinase (MLCK) inhibitor ML-9 in porcine aortic endothelial cells loaded with a Ca2/-sensitive dye fura-2/AM. Bradykinin (10nM) induced a rapid increase in the fura-2 ratio, from basal levels of 1.14{0.09 (mean{SD, nÅ14) to a maximum of 4.16{0.46 in 90sec, followed by a sustained increase (2.58{0.57, 10 min after the addition of bradykinin) (Fig. 1A). The rapid increase in [Ca2/]i was dependent on both intracellular and extracellular Ca2/, while the sustained increase was dependent mainly on the presence of extracellular Ca2/, because bradykinin caused only a small and transient increase in [Ca2/]i in the absence of extracellular Ca2/ (Fig. 1B). Treatment of the cells with either genistein (100mM) or ML-9 (100mM) did not affect the basal [Ca2/]i as compared with control (control: 1.14{0.09 vs. genistein: 1.08{0.02 and ML-9: 1.09{0.04; nÅ14) (Fig.1A). However, pretreatment of the cells with genistein attenuated the bradykinin-stimulated increase in [Ca2/]i in a dose dependent manner i.e., by 48 %, 29% and 19% with 100mM, 50mM, and 10mM genistein, respectively (data not shown). The maximum effect of genistein to inhibit the increase in [Ca2/]i seemed to be attained from 100mM genistein, because no significant difference was observed between 100mM and 500mM (peak value; 3.01{0.18 with 100mM vs 3.07{0.63 with 500mM, N.S.). Pretreatment of endothelial cells with ML-9 (100mM) inhibited the bradykinin-stimulated increase of [Ca2/]i (1.99{0.31 in 30 s after the addition of bradykinin, nÅ14), and reduced it to basal levels 5 min after the addition of bradykinin. On the other hand, genistein (100mM) and ML-9 (100mM) did not affect the bradykinin-stimulated transient rise of [Ca2/]i in the absence of extracellular Ca2/ (Fig. 1B). These findings indicate that genistein and ML-9 attenuate Ca2/ entry from the extracellular space, but do not affect the mobilization of Ca2/ from intracellular stores. ML-9 appears to be more potent in the inhibition of Ca2/ entry from the extracellular space than genistein. Effect of genistein and ML-9 on thapsigargin-stimulated Ca2/ response. The effect of genistein on thapsi-
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FIG. 2. Effect of genistein and ML-9 on thapsigargin-stimulated Ca2/ response. Fura-2/AM loaded cells were pretreated with either genistein (100mM,closed circles) or ML-9 (100mM,open circles) for 10min prior to the application of thapsigargin (1mM) in the presence of 1mM extracellular Ca2/. Closed squares represents treatment with thapsigargin (1mM) alone. Genistein and ML-9 inhibited thapsigargin-stimulated Ca2/ response 2.5min after the administration of thapsigargin, põ0.01. Values are means{SD; nÅ14 cells from 3 separate experiments.
FIG. 1. Effects of genistein and ML-9 on bradykinin-stimulated Ca2/ response in the presence or absence of extracellular Ca2/ in endothelial cells. (A) Cells loaded with fura-2/AM were pretreated for 10min with either genistein (100mM,closed circles) or ML-9 (100mM,open circles) prior to the administration of bradykinin in the presence of 1mM extracellular Ca2/. Closed squares represents treatment with bradykinin (10nM) alone. Genistein and ML-9 attenuated bradykinin-stimulated Ca2/ response 1min after the addition of bradykinin, põ0.01 by student’s t test. Values are means{SD; nÅ14 cells from 3 separate experiments. (B) Extracellular Ca2/-free solution containing 1mM EGTA was introduced 2min before the measurement. Cells were treated for 10min with either genistein (100mM,closed circles) and ML-9 (100mM,open circles) before the application of bradykinin (10nM) in a Ca2/-free solution. Open triangles represents treatment with bradykinin (10nM) alone, põ0.01. Values are means{SD; nÅ14 cells from 3 separate experiments.
gargin-stimulated Ca2/ response was contrasted with the effect of ML-9. Thapsigargin (1mM), an inhibitor of Ca2/-ATPase in the endoplasmic reticulum, induced a slightly delayed but long lasting increase in the fura2 ratio, from basal levels of 0.95{0.15 (mean{SD, nÅ14) to a maximum of 4.23{0.46 in 4 min, followed by a sustained increase (4.21{0.46, 10 min after the addition of thapsigargin, nÅ14) (Fig. 2). Treatment of the cells with either genistein (100mM) or ML-9 (100mM) did not affect the basal [Ca2/]i as compared with control in the presence of extracellular Ca2/ (control: 0.95{0.15 vs. genistein: 1.05{0.15 and ML-9: 1.07{0.08; nÅ14, N.S.) (Fig.2). Pretreatment of the cells with genistein attenuated the thapsigargin-stimulated increase in [Ca2/]i (peak value in the cells with genistein 3.32{0.59 vs 4.23{0.46 in the cells without genistein, nÅ14, põ0.01) . Pretreatment of cells with ML-9 (100mM) inhibited the thapsigargin-stimulated increase of [Ca2/]i (peak value in the cells with ML-9 1.85{0.20 vs 4.23{0.46 in the cells without ML-9, nÅ14, põ0.01), and reduced it to basal levels 8 min after the addition of thapsigargin. These findings indicate that genistein and ML-9 also attenuate Ca2/ entry from the extracellular space, following the stimulation by a receptor independent agonist, thapsigargin. ML9 is more potent in the inhibition of Ca2/ entry in thapsigargin-stimulated cells. Effect of ML-9 on the genistein-insensitive component of Ca2/ influx in bradykinin-stimulated cells. To clarify whether ML-9 could inhibit the genistein-insensitive component of Ca2/ influx in bradykinin-stimulated
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cells, two types of experiments were conducted as following; 1) cells were initially pretreated with ML-9, then treated with genistein after the washout of ML-9 in the continuous presence of bradykinin, and 2) cells were initially pretreated with genistein, then treated with ML-9 after the washout of genistein in the continuous presence of bradykinin. Bradykinin caused only a small and transient rise in [Ca2/]i when the cells were pretreated with ML-9 as shown in Fig.3A, however, the replacement of ML-9 with genistein in the presence of bradykinin revealed the rise in [Ca2/]i again (peak value: ML-9; 1.95{0.01, nÅ14, genistein; 2.75{0.50, nÅ14, põ0.01) (Fig.3A), indicating that genistein failed to inhibit the ML-9 sensitive Ca2/ influx in endothelial cells. In contrast, when the cells were treated with ML9 after the washout of genistein, a further reduction in [Ca2/]i was observed in the bradykinin-stimulated cells (genistein alone; 2.59{0.37, nÅ14 vs 1.89{0.40, nÅ14, põ0.01 in 4 min after the replacement to ML-9) (Fig.3B). These results strongly suggested that the MLCK inhibitor inhibits Ca2/ influx including the tyrosine kinase-insensitive component in bradykinin-stimulated cells. Effect of genistein and ML-9 on bradykinin-stimulated Mn2/ influx. Under the control condition in the presence of extracellular Mn2/(1mM), the fluorescence intensity excited at 360 nm declined gradually to 98{11 % of its initial value in 5 min (Fig.4A, open squares). This may be attributed principally to the passive leakage of Mn2/ into the cells, or to the bleaching of the fluorescence of fura-2. Treatment of the cells with bradykinin significantly reduced the intensity of fluorescence excited by 360 nm to 19{89 % of its initial value in 5 min (Fig.4A, closed squares). This result indicates that bradykinin enhances the influx of Mn2/ as well as Ca2/ into endothelial cells. Pretreatment of the cells with genistein (100mM) did not affect the basal fluorescence intensity at 360nm compared with control, however, it significantly prevented the bradykinin-induced reduction in the fluorescence intensity (49{23% at 5 min after the addition of bradykinin), suggesting the inhibition of Mn2/ influx (Fig.4A, closed circles). Pretreatment of the cells with ML-9 (100mM) also attenuated the bradykinin-induced reduction of the fluorescence intensity at 360 nm without affecting the basal fluorescence (34{14% in 5 min after the addition of bradykinin) (Fig.4A, open circles). These results suggest that the Mn2/ influx in bradykinin-stimulated cells is also attenuated by genistein and ML-9, however, in contrast to the effects on Ca2/ influx, the effect of ML-9 on Mn2/ influx is less potent than that of genistein. DISCUSSION The purpose of the present study was to evaluate which kinase, MLCK or tyrosine kinase could be more
FIG. 3. Effect of ML-9 on the genistein-insensitive component of Ca2/ influx in agonist-stimulated endothelial cells. (A) Cells were pretreated with ML-9 (100mM) for 10min prior to the application of bradykinin (10nM). The cells represented by closed circles were then treated with genistein (100mM) following the washout of ML-9, while the cells represented by open circles were continuously treated with ML-9. A small and transient rise in [Ca2/]i was observed during the stimulation with bradykinin, however, [Ca2/]i increased again in the cells treated with genistein after the washout of ML-9, põ0.01. Values are means{SD; nÅ14 cells from 3 separate experiments. (B) Cells were pretreated with genistein (100mM) for 10min prior to the application of bradykinin (10nM). The cells represented by closed circles were then treated with ML-9 (100mM) after the washout of genistein, while the cells represented by open circles were continuously treated with genistein. Bradykinin caused a rapid increase in [Ca2/]i in the presence of genistein, however, [Ca2/]i was reduced following the replacement of genistein with ML-9, põ0.01. Values are means{SD; nÅ14 cells from 3 separate experiments.
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FIG. 4. Effect of genistein and ML-9 on Mn2/ influx in bradykinin-stimulated endothelial cells. (A) The intensity of fluorescence was expressed as % of initial fluorescence in the fura-2 loaded cells excited by 360nm. Under control conditions, cells (open squares) showed a gradual decrease in the intensity of fluorescence in the presence of 1mM extracellular Mn2/. Bradykinin (10nM) caused a significant reduction in the intensity of fluorescence (closed squares), while treatment with either genistein (100mM, closed circles) or ML9 (100mM, open circles) attenuated the reduction of the fluorescence caused by bradykinin, põ0.01. Values are means{SD; nÅ14 cells from 3 separate experiments. (B) The bars represent % of bradykininstimulated Mn2/ influx in the cells treated with either genistein (100mM) or ML-9 (100mM) 5min after the application of bradykinin. Genistein reduced bradykinin-stimulated Mn2/ influx by 37%, while ML-9 attenuated it by 18%. *põ0.01, **põ0.05. Values are means{SD; nÅ14 cells from 3 separate experiments.
potent and critical in conveying the signal from the intracellular stores to the plasma membrane to initiate Ca2/ influx in agonist-stimulated porcine aortic endothelial cells, and clarify whether it would be also the
case with other divalent cation influx into the cells. We demonstrated that the myosin light chain kinase inhibitor, ML-9 almost completely inhibited bradykinin-and thapsigargin-stimulated Ca2/ influx (9), and the tyrosine kinase inhibitor, genistein only partially inhibited the Ca2/-influx, while neither of these inhibitors affected the mobilization of Ca2/ from intracellular stores. The increase of [Ca2/]i was observed when genistein, in the presence of bradykinin, was added to the cells following the washout of ML-9. In contrast treatment with ML-9 following the washout of genistein significantly reduced the increase in [Ca2/]i in bradykininstimulated endothelial cells. These findings strongly indicate that MLCK plays more critical role than tyrosine kinase in the regulation of Ca2/ influx in agoniststimulated endothelial cells. The pathway for Ca2/-influx has been suggested through a cation channel, because agonists such as bradykinin and histamine stimulate the influx of divalent cations including Mn2/, and Ba2/ as well as Ca2/ (17,24,25). We previously reported that thapsigargin enhanced Mn2/ influx as well as Ca2/ influx in endothelial cells (29). Because of the desirable properties of Mn2/ i.e., the ion (i) quenches fura-2 fluorescence and (ii) has no endogenous agonist-releasable store sites, this ion has been used as an indicator of divalent cation entry when exciting at the isobestic wavelength of 360 nm where the fluorescence is independent of [Ca2/]i (26,27) In the present study bradykinin reduced the fluorescence of fura-2 by 86 % in 6 min, suggesting the stimulated entry of Mn2/. Pretreatment of the cells with genistein attenuated the reduction in the fluorescence by 48 %, and pretreatment of ML-9 attenuated it by 14 % (Fig. 4B). In contrast to Ca2/ influx, genistein inhibited Mn2/ influx more than ML-9 in bradykininstimulated cells. In other words, the MLCK inhibitor is very selective for Ca2/ as opposed to Mn2/. Although non-muscle cells including neutrophils (24), mast cells (29) and 3T3 cells (30,31) have been shown to possess non-selective cation channels which promote receptormediated Ca2/ influx, these results may suggest that Mn2/ may not follow an identical route to that of receptor-mediated Ca2/ influx. This reservation might be a possibility in endothelial cells given the observation by Jacob that stimulation by a low dose of histamine stimulates Mn2/ to enter the cytoplasm in an apparently non-pulsatile manner while [Ca2/]i is repetitively spiking (14,16,32). In conclusion, we have demonstrated that (i) inhibition of MLCK almost completely abolishes the Ca2/ influx in bradykinin-stimulated endothelial cells including the tyrosine kinase inhibitor insensitive component, and (ii) the MLCK inhibitor is less potent in the inhibition of Mn2/ influx than the tyrosine kinase inhibitor. Our data indicate the critical role of MLCK in the regulation of Ca2/ influx, and suggest a different
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selectivity for Ca2/ and Mn2/ in the cation entry pathway in agonist-stimulated endothelial cells. ACKNOWLEDGMENTS We express our thanks Yoriko Hirade for her technical assistance and Dr. Cherie Millar for critical reading of the manuscript. This study was supported by Ministry of Education, Culture, and Science of Japan Grants-in-Aid 07670772 and Japan Foundation of Cardiovascular Research to H. Watanabe.
REFERENCES 1. Furchgott, R. F., and Zawadzki, J. V. (1980) Nature 228, 373– 376. 2. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yasaki, Y., Goto, K., and Masaki, T. (1988) Nature 332, 411–415. 3. Schweigerer, L., Neufeld, G., Freideman, J., Abraham, J. A., Feddes, J. C., and Gospodarowicz, D. (1987) Nature 325, 257– 259. 4. Mendelowitzz, D., Bacal, K., and Kunze, D. L. (1992) Am. J. Physiol. 262, H942–H948. 5. Freay, A., Johns, A., Adams, D. J., Ryan, U. S., and Breemen, C. V. (1989) Pflu¨gers Arch. 414, 377–384. 6. Berridge, M. J. (1993) Nature 361, 315–325. 7. Tsien, R. W., and Tsien, R. Y. (1990) Annu. Rev. Cell Biol. 6, 715–760. 8. Putney, J. W. Jr., and Bird, G. ST. J. (1993) Endocrine Rev. 14, 610–631. 9. Watanabe, H., Takahashi, R., Zhang, X., Kakizawa, H., Hayashi, H., and Ohno, R. (1996) Biochem. Biophys. Res. Commun. (1996) 225, 777–784. 10. Fleming, I., Beate, F., and Rudi, B. (1995) Circ. Res. 76, 522– 529. 11. Hans-J, K., Emil, V. N., Peter, C. W., and Wolfgang Siess. (1994) Biochem. Biophys. Res. Commun. 202, 1651–1656. 12. Murphy, C. T., Poll, C. T., and Weswick, J. (1995) Cell Calcium 18, 245–251.
13. Sugden, P. H., and Bogoyevitch, M. A. (1995) Cardiovasc.Res. 30, 478–492. 14. Hallen, T. J., Jacob, R., and Merritt, J. E. (1988) Biochem. J. 255, 179–184. 15. Hallen, T. J., Jacob, R., and Merritt, J. E. (1990) J. Physiol. 421, 55–77. 16. Jacob, R. (1990) J. Physiol. 421, 55–77. 17. Yamamoto, N., Watanabe, H., Kakizawa, H., Hirano, M., Kobayashi, A., and Ohno, R. (1995) Biochim. Biopys. Acta. 1266, 157– 162. 18. Colden-Stanfield, M., Schlling, W. P., Ritchie, A. K., Eskin, S. G., Navarro, L. T., and Kunze, D. L. (1987) Circ. Res. 61, 632–640. 19. Cannell, M. B., and Sage, S. O. (1989) J.Physiol. (Lond.) 419, 555–568. 20. Noll, T., Muhs, A., Besselmann, M., Watanabe, H., and Piper, H. M. (1995) Am. J. Physiol. 268, H1462–H1470. 21. Watanabe, H., Kuhne, W., Spahr, R., Schwartz, P., and Piper, H. M. (1991) Am. J. Physiol. 260, H1344–H1352. 22. Li, Q., Altschuld, R. A., and Stokes, B. T., (1987) Biochem. Biophys. Res. Commun. 147, 120–126. 23. Grynkiewicsz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol.Chem. 260, 3440–3450. 24. Bassam, B. D., Shaun, R. M., Kuldeep, N., Caloline, A. H., and Paul, H. N. (1996) J. Biol. Chem. 271, 20540–20544. 25. Joseph, H. S., and William, P. D. (1994) Am. J. Physiol. 266, G475–G484. 26. Heketh, R. T., Smith, G. A., Moore, J. P., Taylor, M. V., and Metcalfe, J. C. (1983) J. Biol. Chem. 258, 4876–4882. 27. Tsien, R. Y., Rink, T. J., and Poenie, M. (1985) Cell Calcium 6, 145–157. 28. Merrit, J. E., Jacob, R., and Hallam, T. J. (1989) J. Biol. Chem. 264, 1522–1527. 29. Hirashima, N., and Kirino, Y. (1992) Biochem. Biophys. Res. Commun. 147, 793–799. 30. Huang, B. Y., and Stanley, G. R. (1993) J. Physiol. 461, 601– 618. 31. Kanzaki, M., Shibata, H., Mogami, H., and Kojima, I. (1995) J. Biol. Chem. 270, 13099–13104. 32. Hallen, T. J., Jacob, R., and Merritt, J. E. (1989) Biochem. J. 259, 125–129.
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Roles of Inhibitors of Myosin Light Chain Kinase and Tyrosine Kinase on Cation Influx in Agonist-Stimulated Endothelial Cells Reiko Takahashi,1 Hiroshi Watanabe, Xu-Xia Zhang, Hiroyasu Kakizawa, Hideharu Hayashi, and Ryuzo Ohno Internal Medicine III, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-31, Japan
Received May 6, 1997
Agoinst-stimulated Ca2/ influx is critically important to mediate the function of endothelial cells. It has been suggested that release of Ca2/ from internal stores activates Ca2/ influx across the plasma membrane. In the present study, we investigated the effects of ML-9, a myosin light-chain kinase (MLCK) inhibitor, and genistein, a tyrosine kinase inhibitor, on the agonist stimulated Ca2/ response in porcine aortic endothelial cells loaded with a Ca2/-sensitive dye, fura-2. ML-9 almost completely abolished Ca2/ influx, whereas genistein only partially attenuated Ca2/ entry. Both of them did not affect the mobilization of Ca2/ from internal stores. In contrast, genistein was more potent in the inhibition of Mn2/ influx than ML-9. These findings indicate the different selectivity for Ca2/ and Mn2/ in the cation entry pathway in agonist-stimulated endothelial cells. q 1997 Academic Press
Endothelial cells are multifunctional cells that regulate permeability, between the plasma and the interstitial space, vascular tone, by releasing endothelial derived relaxing and/or constricting factors, and are involved in angiogenesis, anticoagulation and inflammatory responses (1-3). The cytosolic calcium ion is one of the major second messengers that mediate the functions of endothelial cells. The intracellular concentration of Ca2/([Ca2/]i) shows biphasic increases in response to agonist stimulation with a rapid initial increase followed by a sustained increase. The initial increase [Ca2/]i is largely dependent on the release of calcium from Inositol 1,4,5-Triphosphate (Ins(1,4,5)P3)sensitive stores (4,5). The sustained increase in [Ca2/]i 1 To whom correspondence should be addressed. Fax: /81 53 434 2910. E-mail: [email protected]. Abbreviations used: MLC, myosin light-chain; MLCK, myosin light-chain kinase; Ins(1,4,5)P3 , inositol 1,4,5-triphosphate.
is caused by Ca2/ influx from the extracellular space. Although it has been suggested that the influx of Ca2/ can be triggered by the depletion of internal Ca2/ stores (6-8), the mechanism(s) communicating the release of Ca2/ from intracellular stores with the influx of Ca2/ from extracellular space is not well understood. Recently we reported that myosin light-chain kinase (MLCK) inhibitors, ML-9 and wortmannin inhibited Ca2/ influx from the extracellular space without affecting the release of Ca2/ from internal stores in bradykinin- and thapsigargin-treated porcine aortic endothelial cells (9). The data indicate that a common part of the signal transduction cascade initiated by a receptordependent and -independent stimulation of endothelial cells includes a MLCK inhibitor-sensitive influx of Ca2/ across the plasma membrane. On the other hand, it has been shown that inhibition of protein tyrosine phosphorylation by different tyrosine kinase inhibitors, herbimycin A, genistein and piceatannol attenuated agonist-stimulated Ca2/ entry in venous endothelial cells from human umbilical cords (10,11). However, the potency these inhibitors to inhibit the Ca2/ influx was not clarified in agonist-stimulated endothelial cells (12,13). Therefore, one of the purposes of this study is to determine which kinase inhibitor plays the major role in the regulation of the influx of Ca2/ in endothelial cells. Agonist stimulation has been shown to enhance not only Ca2/ influx but also Mn2/ influx into the cells in the presence of extracellular Mn2/. Mn2/ influx is assessed by the quenching of the fluorescence of fura-2 (14-16). The pathway for this cation entry remains to be determined. In our previous study, the influx of Ca2/ caused by a receptor-independent agonist thapsigargin was attenuated by either membrane depolarization due to a high K/ solution or the pretreatment of endothelial cells with NiCl2 , but was not influenced by verapamil a blocking agent of voltage dependent Ca2/ channel nor the replacement of Na/ by Li/ in the extracellular
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solution (17). These results suggest the involvement of non-specific cation channels independent of the voltage-dependent Ca2/ channels and/or Na//Ca2/ exchange mechanisms (18,19). Another purpose of this study is to evaluate whether inhibitors of MLCK and tyrosine kinase can modify the influx of other cations such as Mn2/ across the plasma membrane. We report in the present study that the MLCK inhibitor, ML-9 almost completely inhibits the Ca2/ influx including tyrosine kinase inhibitor-insensitive component in bradykinin-stimulated endothelial cells, suggesting that MLCK is potentially more important than tyrosine kinase in the regulation of Ca2/ influx in endothelial cells. In contrast, tyrosine kinase inhibitor, genistein is more potent in the inhibition of Mn2/ influx than the MLCK inhibitor. These findings indicate the different selectivity for Ca2/ and Mn2/ in the cation entry pathway in agonist-stimulated endothelial cells. MATERIALS AND METHODS Cell culture. Porcine aortic endothelial cells were isolated, as previously described (20,21) by gentle mechanical scraping the intima of the descending part of porcine aortas. After centrifugation at 250 1 g for 10 min in M199 solution, the fraction of endothelial cells was purified from this suspension, resuspended in M199 solution with Earle’s salts, supplemented 100 IU/ml penicillin G, 100 mg/ml streptomycin, and 20 % newborn calf serum (NCS), then aliquotted into the polybiphenyl dishes fixed on 10 1 10-mm glass cover slips, and cultured in an incubator at 377C under 5% CO2 for two days. The medium was renewed every day. Measurement of cytosolic calcium concentration. [Ca2/]i was measured, as previously described (17) in endothelial cells adhering to the glass cover slip. The cells were incubated for 45 minutes in modified Tyrode’s solution (composition in mM: 150.0 NaCl, 2.7 KCl, 1.2 KH2PO4 , 1.2 MgSO4 , 1.0 CaCl2 , and 10.0 N-2-hydroxyethyl-piperazine-N*-2-ethanesulfonic acid, with pH 7.4 at 257C) containing 10% NCS and 2mM fura-2/AM, a fluorescent Ca2/ indicator. The cells were subsequently washed three times with modified Tyrode’s solution to remove the fura-2/AM and the serum from the extracellular fluid followed by a 20-min postincubation period before measurements were started. All experiments were performed at 257C. The absorption shift of fura-2 that occurs upon binding can be determined by scanning the excitation spectrum between 340 and 380 nm while monitoring the emission at 510 nm. The fluorescent image was analyzed every 30 s from the individual cells with an [Ca2/]i analyzer (Argus 50, Hamamatsu Photonics) using a ultra-high sensitivity television camera (CCD). The fluorescence ratio (F340/ F380) was obtained by dividing, pixel by pixel, the 340-nm image after background subtraction by the 380-nm image after background subtraction. Intracellular calibration was performed in accordance with the method of Li et al. (22). In order to obtain the maximum Rmax or minimum Rmin value of the fluorescence ratio, after fura-2 loading, the endothelial cells were exposed to modified Tyrode’s solution containing 10 mM ionomycin and 3 mM of Ca2/ or 5 mM ethylene glycol-bis(ßaminoethyl ether)-N,N,N*,N*-tetraacetic acid (EGTA), respectively. [Ca2/]i was calculated from the equation of Grynkiewicz et al. (23). Bradykinin, thapsigargin, ML-9, and genistein had no effect on fura2 fluorescence itself, or on autofluorescence of unloaded cells when examined at concentrations employed in this study. Measurement of influx of Mn2/. Fura-2 has a higher affinity for Mn2/ (Kd Å 5.33 nM) than for Ca2/ (23). If Mn2/ enters into cells, the binding of Mn2/ with fura-2 quenches its fluorescence. Since the
absorption of fura-2 excited at 360 nm is not affected depending on [Ca2/]i , the decline of fluorescence excited at 360 nm can be utilized to monitor Mn2/ influx into cells (17). Materials. Medium 199, newborn calf serum and penicillin-streptomycin were purchased from GIBCO (New York, USA), fura-2/AM was from Dojindo (Kumamoto, Japan), and from Wako(Osaka, Japan). Bradykinin, thapsigargin, ML-9, and genistein were from Sigma (St. Louis, USA). All other chemicals were of analytical grade.
RESULTS Effect of genistein and ML-9 on bradykinin-stimulated Ca2/ response. The effect of the tyrosine kinase inhibitor genistein on bradykinin-stimulated Ca2/ response was contrasted with the effect of the myosin light chain kinase (MLCK) inhibitor ML-9 in porcine aortic endothelial cells loaded with a Ca2/-sensitive dye fura-2/AM. Bradykinin (10nM) induced a rapid increase in the fura-2 ratio, from basal levels of 1.14{0.09 (mean{SD, nÅ14) to a maximum of 4.16{0.46 in 90sec, followed by a sustained increase (2.58{0.57, 10 min after the addition of bradykinin) (Fig. 1A). The rapid increase in [Ca2/]i was dependent on both intracellular and extracellular Ca2/, while the sustained increase was dependent mainly on the presence of extracellular Ca2/, because bradykinin caused only a small and transient increase in [Ca2/]i in the absence of extracellular Ca2/ (Fig. 1B). Treatment of the cells with either genistein (100mM) or ML-9 (100mM) did not affect the basal [Ca2/]i as compared with control (control: 1.14{0.09 vs. genistein: 1.08{0.02 and ML-9: 1.09{0.04; nÅ14) (Fig.1A). However, pretreatment of the cells with genistein attenuated the bradykinin-stimulated increase in [Ca2/]i in a dose dependent manner i.e., by 48 %, 29% and 19% with 100mM, 50mM, and 10mM genistein, respectively (data not shown). The maximum effect of genistein to inhibit the increase in [Ca2/]i seemed to be attained from 100mM genistein, because no significant difference was observed between 100mM and 500mM (peak value; 3.01{0.18 with 100mM vs 3.07{0.63 with 500mM, N.S.). Pretreatment of endothelial cells with ML-9 (100mM) inhibited the bradykinin-stimulated increase of [Ca2/]i (1.99{0.31 in 30 s after the addition of bradykinin, nÅ14), and reduced it to basal levels 5 min after the addition of bradykinin. On the other hand, genistein (100mM) and ML-9 (100mM) did not affect the bradykinin-stimulated transient rise of [Ca2/]i in the absence of extracellular Ca2/ (Fig. 1B). These findings indicate that genistein and ML-9 attenuate Ca2/ entry from the extracellular space, but do not affect the mobilization of Ca2/ from intracellular stores. ML-9 appears to be more potent in the inhibition of Ca2/ entry from the extracellular space than genistein. Effect of genistein and ML-9 on thapsigargin-stimulated Ca2/ response. The effect of genistein on thapsi-
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FIG. 2. Effect of genistein and ML-9 on thapsigargin-stimulated Ca2/ response. Fura-2/AM loaded cells were pretreated with either genistein (100mM,closed circles) or ML-9 (100mM,open circles) for 10min prior to the application of thapsigargin (1mM) in the presence of 1mM extracellular Ca2/. Closed squares represents treatment with thapsigargin (1mM) alone. Genistein and ML-9 inhibited thapsigargin-stimulated Ca2/ response 2.5min after the administration of thapsigargin, põ0.01. Values are means{SD; nÅ14 cells from 3 separate experiments.
FIG. 1. Effects of genistein and ML-9 on bradykinin-stimulated Ca2/ response in the presence or absence of extracellular Ca2/ in endothelial cells. (A) Cells loaded with fura-2/AM were pretreated for 10min with either genistein (100mM,closed circles) or ML-9 (100mM,open circles) prior to the administration of bradykinin in the presence of 1mM extracellular Ca2/. Closed squares represents treatment with bradykinin (10nM) alone. Genistein and ML-9 attenuated bradykinin-stimulated Ca2/ response 1min after the addition of bradykinin, põ0.01 by student’s t test. Values are means{SD; nÅ14 cells from 3 separate experiments. (B) Extracellular Ca2/-free solution containing 1mM EGTA was introduced 2min before the measurement. Cells were treated for 10min with either genistein (100mM,closed circles) and ML-9 (100mM,open circles) before the application of bradykinin (10nM) in a Ca2/-free solution. Open triangles represents treatment with bradykinin (10nM) alone, põ0.01. Values are means{SD; nÅ14 cells from 3 separate experiments.
gargin-stimulated Ca2/ response was contrasted with the effect of ML-9. Thapsigargin (1mM), an inhibitor of Ca2/-ATPase in the endoplasmic reticulum, induced a slightly delayed but long lasting increase in the fura2 ratio, from basal levels of 0.95{0.15 (mean{SD, nÅ14) to a maximum of 4.23{0.46 in 4 min, followed by a sustained increase (4.21{0.46, 10 min after the addition of thapsigargin, nÅ14) (Fig. 2). Treatment of the cells with either genistein (100mM) or ML-9 (100mM) did not affect the basal [Ca2/]i as compared with control in the presence of extracellular Ca2/ (control: 0.95{0.15 vs. genistein: 1.05{0.15 and ML-9: 1.07{0.08; nÅ14, N.S.) (Fig.2). Pretreatment of the cells with genistein attenuated the thapsigargin-stimulated increase in [Ca2/]i (peak value in the cells with genistein 3.32{0.59 vs 4.23{0.46 in the cells without genistein, nÅ14, põ0.01) . Pretreatment of cells with ML-9 (100mM) inhibited the thapsigargin-stimulated increase of [Ca2/]i (peak value in the cells with ML-9 1.85{0.20 vs 4.23{0.46 in the cells without ML-9, nÅ14, põ0.01), and reduced it to basal levels 8 min after the addition of thapsigargin. These findings indicate that genistein and ML-9 also attenuate Ca2/ entry from the extracellular space, following the stimulation by a receptor independent agonist, thapsigargin. ML9 is more potent in the inhibition of Ca2/ entry in thapsigargin-stimulated cells. Effect of ML-9 on the genistein-insensitive component of Ca2/ influx in bradykinin-stimulated cells. To clarify whether ML-9 could inhibit the genistein-insensitive component of Ca2/ influx in bradykinin-stimulated
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cells, two types of experiments were conducted as following; 1) cells were initially pretreated with ML-9, then treated with genistein after the washout of ML-9 in the continuous presence of bradykinin, and 2) cells were initially pretreated with genistein, then treated with ML-9 after the washout of genistein in the continuous presence of bradykinin. Bradykinin caused only a small and transient rise in [Ca2/]i when the cells were pretreated with ML-9 as shown in Fig.3A, however, the replacement of ML-9 with genistein in the presence of bradykinin revealed the rise in [Ca2/]i again (peak value: ML-9; 1.95{0.01, nÅ14, genistein; 2.75{0.50, nÅ14, põ0.01) (Fig.3A), indicating that genistein failed to inhibit the ML-9 sensitive Ca2/ influx in endothelial cells. In contrast, when the cells were treated with ML9 after the washout of genistein, a further reduction in [Ca2/]i was observed in the bradykinin-stimulated cells (genistein alone; 2.59{0.37, nÅ14 vs 1.89{0.40, nÅ14, põ0.01 in 4 min after the replacement to ML-9) (Fig.3B). These results strongly suggested that the MLCK inhibitor inhibits Ca2/ influx including the tyrosine kinase-insensitive component in bradykinin-stimulated cells. Effect of genistein and ML-9 on bradykinin-stimulated Mn2/ influx. Under the control condition in the presence of extracellular Mn2/(1mM), the fluorescence intensity excited at 360 nm declined gradually to 98{11 % of its initial value in 5 min (Fig.4A, open squares). This may be attributed principally to the passive leakage of Mn2/ into the cells, or to the bleaching of the fluorescence of fura-2. Treatment of the cells with bradykinin significantly reduced the intensity of fluorescence excited by 360 nm to 19{89 % of its initial value in 5 min (Fig.4A, closed squares). This result indicates that bradykinin enhances the influx of Mn2/ as well as Ca2/ into endothelial cells. Pretreatment of the cells with genistein (100mM) did not affect the basal fluorescence intensity at 360nm compared with control, however, it significantly prevented the bradykinin-induced reduction in the fluorescence intensity (49{23% at 5 min after the addition of bradykinin), suggesting the inhibition of Mn2/ influx (Fig.4A, closed circles). Pretreatment of the cells with ML-9 (100mM) also attenuated the bradykinin-induced reduction of the fluorescence intensity at 360 nm without affecting the basal fluorescence (34{14% in 5 min after the addition of bradykinin) (Fig.4A, open circles). These results suggest that the Mn2/ influx in bradykinin-stimulated cells is also attenuated by genistein and ML-9, however, in contrast to the effects on Ca2/ influx, the effect of ML-9 on Mn2/ influx is less potent than that of genistein. DISCUSSION The purpose of the present study was to evaluate which kinase, MLCK or tyrosine kinase could be more
FIG. 3. Effect of ML-9 on the genistein-insensitive component of Ca2/ influx in agonist-stimulated endothelial cells. (A) Cells were pretreated with ML-9 (100mM) for 10min prior to the application of bradykinin (10nM). The cells represented by closed circles were then treated with genistein (100mM) following the washout of ML-9, while the cells represented by open circles were continuously treated with ML-9. A small and transient rise in [Ca2/]i was observed during the stimulation with bradykinin, however, [Ca2/]i increased again in the cells treated with genistein after the washout of ML-9, põ0.01. Values are means{SD; nÅ14 cells from 3 separate experiments. (B) Cells were pretreated with genistein (100mM) for 10min prior to the application of bradykinin (10nM). The cells represented by closed circles were then treated with ML-9 (100mM) after the washout of genistein, while the cells represented by open circles were continuously treated with genistein. Bradykinin caused a rapid increase in [Ca2/]i in the presence of genistein, however, [Ca2/]i was reduced following the replacement of genistein with ML-9, põ0.01. Values are means{SD; nÅ14 cells from 3 separate experiments.
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FIG. 4. Effect of genistein and ML-9 on Mn2/ influx in bradykinin-stimulated endothelial cells. (A) The intensity of fluorescence was expressed as % of initial fluorescence in the fura-2 loaded cells excited by 360nm. Under control conditions, cells (open squares) showed a gradual decrease in the intensity of fluorescence in the presence of 1mM extracellular Mn2/. Bradykinin (10nM) caused a significant reduction in the intensity of fluorescence (closed squares), while treatment with either genistein (100mM, closed circles) or ML9 (100mM, open circles) attenuated the reduction of the fluorescence caused by bradykinin, põ0.01. Values are means{SD; nÅ14 cells from 3 separate experiments. (B) The bars represent % of bradykininstimulated Mn2/ influx in the cells treated with either genistein (100mM) or ML-9 (100mM) 5min after the application of bradykinin. Genistein reduced bradykinin-stimulated Mn2/ influx by 37%, while ML-9 attenuated it by 18%. *põ0.01, **põ0.05. Values are means{SD; nÅ14 cells from 3 separate experiments.
potent and critical in conveying the signal from the intracellular stores to the plasma membrane to initiate Ca2/ influx in agonist-stimulated porcine aortic endothelial cells, and clarify whether it would be also the
case with other divalent cation influx into the cells. We demonstrated that the myosin light chain kinase inhibitor, ML-9 almost completely inhibited bradykinin-and thapsigargin-stimulated Ca2/ influx (9), and the tyrosine kinase inhibitor, genistein only partially inhibited the Ca2/-influx, while neither of these inhibitors affected the mobilization of Ca2/ from intracellular stores. The increase of [Ca2/]i was observed when genistein, in the presence of bradykinin, was added to the cells following the washout of ML-9. In contrast treatment with ML-9 following the washout of genistein significantly reduced the increase in [Ca2/]i in bradykininstimulated endothelial cells. These findings strongly indicate that MLCK plays more critical role than tyrosine kinase in the regulation of Ca2/ influx in agoniststimulated endothelial cells. The pathway for Ca2/-influx has been suggested through a cation channel, because agonists such as bradykinin and histamine stimulate the influx of divalent cations including Mn2/, and Ba2/ as well as Ca2/ (17,24,25). We previously reported that thapsigargin enhanced Mn2/ influx as well as Ca2/ influx in endothelial cells (29). Because of the desirable properties of Mn2/ i.e., the ion (i) quenches fura-2 fluorescence and (ii) has no endogenous agonist-releasable store sites, this ion has been used as an indicator of divalent cation entry when exciting at the isobestic wavelength of 360 nm where the fluorescence is independent of [Ca2/]i (26,27) In the present study bradykinin reduced the fluorescence of fura-2 by 86 % in 6 min, suggesting the stimulated entry of Mn2/. Pretreatment of the cells with genistein attenuated the reduction in the fluorescence by 48 %, and pretreatment of ML-9 attenuated it by 14 % (Fig. 4B). In contrast to Ca2/ influx, genistein inhibited Mn2/ influx more than ML-9 in bradykininstimulated cells. In other words, the MLCK inhibitor is very selective for Ca2/ as opposed to Mn2/. Although non-muscle cells including neutrophils (24), mast cells (29) and 3T3 cells (30,31) have been shown to possess non-selective cation channels which promote receptormediated Ca2/ influx, these results may suggest that Mn2/ may not follow an identical route to that of receptor-mediated Ca2/ influx. This reservation might be a possibility in endothelial cells given the observation by Jacob that stimulation by a low dose of histamine stimulates Mn2/ to enter the cytoplasm in an apparently non-pulsatile manner while [Ca2/]i is repetitively spiking (14,16,32). In conclusion, we have demonstrated that (i) inhibition of MLCK almost completely abolishes the Ca2/ influx in bradykinin-stimulated endothelial cells including the tyrosine kinase inhibitor insensitive component, and (ii) the MLCK inhibitor is less potent in the inhibition of Mn2/ influx than the tyrosine kinase inhibitor. Our data indicate the critical role of MLCK in the regulation of Ca2/ influx, and suggest a different
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selectivity for Ca2/ and Mn2/ in the cation entry pathway in agonist-stimulated endothelial cells. ACKNOWLEDGMENTS We express our thanks Yoriko Hirade for her technical assistance and Dr. Cherie Millar for critical reading of the manuscript. This study was supported by Ministry of Education, Culture, and Science of Japan Grants-in-Aid 07670772 and Japan Foundation of Cardiovascular Research to H. Watanabe.
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13. Sugden, P. H., and Bogoyevitch, M. A. (1995) Cardiovasc.Res. 30, 478–492. 14. Hallen, T. J., Jacob, R., and Merritt, J. E. (1988) Biochem. J. 255, 179–184. 15. Hallen, T. J., Jacob, R., and Merritt, J. E. (1990) J. Physiol. 421, 55–77. 16. Jacob, R. (1990) J. Physiol. 421, 55–77. 17. Yamamoto, N., Watanabe, H., Kakizawa, H., Hirano, M., Kobayashi, A., and Ohno, R. (1995) Biochim. Biopys. Acta. 1266, 157– 162. 18. Colden-Stanfield, M., Schlling, W. P., Ritchie, A. K., Eskin, S. G., Navarro, L. T., and Kunze, D. L. (1987) Circ. Res. 61, 632–640. 19. Cannell, M. B., and Sage, S. O. (1989) J.Physiol. (Lond.) 419, 555–568. 20. Noll, T., Muhs, A., Besselmann, M., Watanabe, H., and Piper, H. M. (1995) Am. J. Physiol. 268, H1462–H1470. 21. Watanabe, H., Kuhne, W., Spahr, R., Schwartz, P., and Piper, H. M. (1991) Am. J. Physiol. 260, H1344–H1352. 22. Li, Q., Altschuld, R. A., and Stokes, B. T., (1987) Biochem. Biophys. Res. Commun. 147, 120–126. 23. Grynkiewicsz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol.Chem. 260, 3440–3450. 24. Bassam, B. D., Shaun, R. M., Kuldeep, N., Caloline, A. H., and Paul, H. N. (1996) J. Biol. Chem. 271, 20540–20544. 25. Joseph, H. S., and William, P. D. (1994) Am. J. Physiol. 266, G475–G484. 26. Heketh, R. T., Smith, G. A., Moore, J. P., Taylor, M. V., and Metcalfe, J. C. (1983) J. Biol. Chem. 258, 4876–4882. 27. Tsien, R. Y., Rink, T. J., and Poenie, M. (1985) Cell Calcium 6, 145–157. 28. Merrit, J. E., Jacob, R., and Hallam, T. J. (1989) J. Biol. Chem. 264, 1522–1527. 29. Hirashima, N., and Kirino, Y. (1992) Biochem. Biophys. Res. Commun. 147, 793–799. 30. Huang, B. Y., and Stanley, G. R. (1993) J. Physiol. 461, 601– 618. 31. Kanzaki, M., Shibata, H., Mogami, H., and Kojima, I. (1995) J. Biol. Chem. 270, 13099–13104. 32. Hallen, T. J., Jacob, R., and Merritt, J. E. (1989) Biochem. J. 259, 125–129.
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