Abstract
ML-7, (5-iodonaphthalene-1-sulfonyl) homopiperazine, is commonly employed as a myosin light chain kinase (MLCK) inhibitor. In the present study, we demonstrated that ML-7 affects the superoxide (O2 −)-producing system of human neutrophils in an MLCK-independent manner. Human neutrophils were stimulated with phorbol myristate acetate (PMA), which does not activate MLCK. ML-7 inhibited extracellular release, but not intracellular production of O2 − in the stimulated cells. Fluorescence microscopy revealed the generation of O2 − at intracellular compartments in the stimulated cells exposed to ML-7. At the electron microscopic level, the reaction product of NADPH oxidase activity was found in intracellular compartments. ML-7 strongly inhibited the association of the oxidant-producing intracellular compartments with the plasma membrane. Furthermore, the upregulation of alkaline phosphatase activity, a marker enzyme of the oxidant-producing intracellular compartments, was also inhibited by ML-7. These findings indicate that ML-7 inhibits the fusion of the oxidant-producing intracellular compartments to the plasma membrane resulting in the inhibition of the extracellular release of O2 − in PMA-stimulated human neutrophils in an MLCK-independent manner.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Babior BM (1999) NADPH oxidase: an update. Blood 93:1464–1476
Borregaard N, Cowland JB (1997) Granules in the human neutrophilic polymorphonuclear leukocyte. Blood 89:3503–3521
Briggs RT, Drath DB, Karnovsky ML, Karnovsky MJ (1975) Localization of NADH oxidase on the surface of human polymorphonuclear leukocytes by a new cytochemical method. J Cell Biol 67:566–586
Choi OH, Adelstein RS, Beaven MA (1994) Secretion from rat basophilic RBL-2H3 cells is associated with dephosphorylation of myosin light chains by myosin light chain kinase as well as phosphorylation by protein kinase C. J Biol Chem 269:536–541
Christiansen NO, Larsen CS, Juhl H (1986) Ca2+ and phorbol ester activation of protein kinase C at intracellular Ca2+ concentrations and the effect of TMB-8. Biochim Biophys Acta 882:57–62
Eddy RJ, Pierini LM, Matsumura F, Maxfield FR (2000) Ca2+-dependent myosin II activation is required for uropod retraction during neutrophil migration. J Cell Sci 113:1287–1298
Garcia JG, Verin AD, Herenyiova M, English D (1998) Adherent neutrophils activate endothelial myosin light chain kinase: role in transendothelial migration. J Appl Physiol 84:1817–1821
Gullberg U, Anderson E, Garwicz D, Lindmark A, Olsson I (1997) Biosynthesis, processing and sorting of neutrophil proteins: insight into neutrophil granule development. Eur J Haematol 58:137–153
Heyworth PG, Erickson RW, Ding J, Curnutte JT, Badwey JA (1995) Naphthalene sulphonamides block neutrophil superoxide production by intact cells and in a cell-free system: is myosin light chain kinase responsible for these effects? Biochem J 311:81–87
Heyworth PG, Robinson JM, Ding J, Ellios BA, Badway JA (1997) Cofilin undergoes rapid dephosphorylation in stimulated neutrophils and translocates to ruffled membranes enriched in products of the NADPH oxidase complex. Evidence for a novel cycle of phosphorylation and dephosphorylation. Histochem Cell Biol 108:221–233
Kim MH, Kim SH, Kim HS, Chang JW, Hong YS, Kim HW, Park CS (1998) Regulation of renin secretion through reversible phosphorylation of myosin by myosin light chain kinase and protein phosphatase type 1. J Pharmacol Exp Ther 285:968–974
Kobayashi T, Robinson JM (1991) A novel intracellular compartment with unusual secretory properties in human neutrophils. J Cell Biol 113:743–756
Kobayashi T, Seguchi H (1999) Novel insight into current models of NADPH oxidase regulation, assembly and localization in human polymorphonuclear leukocytes. Histol Histopathol 14:1295–1308
Kobayashi T, Robinson JM, Seguchi H (1998) Identification of intracellular sites of superoxide production in stimulated neutrophils. J Cell Sci 111:81–91
Kobayashi T, Garcia del Saz E, Hendry J, Seguchi H (1999) Detection of oxidant producing-sites in glutaraldehyde-fixed human neutrophils and eosinophils stimulated with phorbol myristate acetate. Histochem J 31:181–194
Kobayashi T, Zinchuk VS, Okada T, Wakiguchi H, Kurashige T, Takatsuji H, Seguchi H (2000) A simple approach for the analysis of intracellular movement of oxidant-producing intracellular compartments in living human neutrophils. Histochem Cell Biol 113:251–257
Kobayashi T, Tsunawaki S, Seguchi H (2001) Evaluation of the process for superoxide production by NADPH oxidase in human neutrophils: evidence for cytoplasmic origin of superoxide. Redox Rep 6:27–36
Leavey PJ, Gonzalez-Aller C, Thurman G, Kleinberg M, Rinckel L, Ambruso DW, Freeman S, Kuypers FA, Ambruso DR (2002) A 29-kDa protein associated with p67phox expresses both peroxiredoxin and phospholipase A2 activity and enhances superoxide anion production by a cell-free system of NADPH oxidase activity. J Biol Chem 277:45181–45187
Mansfield PJ, Shayman JA, Boxer LA (2000) Regulation of polymorphonuclear leukocyte phagocytosis by myosin light chain kinase after activation of mitogen-activated protein kinase. Blood 95:2407–2412
Massey V (1959) The microestimation of succinate and the extinction coefficient of cytochrome c. Biochem Biophys Acta 34:255–256
Matsumura C, Kuwashima H, Kimura T (2000) Lack of Ca2+- and ATP-dependent priming state in caffeine-induced exocytosis in bovine adrenal chromaffin cells: comparison with Ca2+. J Auton Pharmacol 20:31–36
McCord JM, Fridovich I (1969) Superoxide dismutase: an enzymatic function for erythrocuprein (hemocuprein). J Biol Chem 244:6049–6055
Morimatsu T, Kawagoshi A, Yoshida K, Tamura M (1997) Actin enhances activation of human neutrophil NADPH oxidase in a cell-free system. Biochem Biophys Res Commun 230:206–210
Nishizuka Y (1986) Studies and perspectives of protein kinase C. Science 233:305–312
Robinson JP, Bruner LH, Bassoe CF, Hudson JL, Ward PA, Phan SH (1988) Measurement of intracellular fluorescence of human monocytes relative to oxidative metabolism. J Leukoc Biol 43:304–310
Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31–43
Staniek K, Gille L, Kozlov AV, Nohl H (2002) Mitochondrial superoxide radical formation is controlled by electron bifurcation to the high and low potential pathways. Free Radic Res 36:381–387
St-Pierre J, Buckingham JA, Roebuck SJ, Brand MD (2002) Topology of superoxide production from different sites in the mitochondrial electron transport chain. J Biol Chem 277:44784–44790
Totsukawa G, Yamakita Y, Yamashiro S, Hartshorne DJ, Yasuharu S, Matsumura F (2000) Distinct roles of ROCK(Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts. J Cell Biol 150:797–806
Yamamoto M, Okimasu E, Terada S, Utsumi K (1987) Mechanism of polymorphonuclear leukocyte activation by myristate. Involvement of calcium ion and protein kinase C. Cell Struct Funct 12:357–367
Acknowledgement
We are grateful to Dr. Pamela J. Mansfield (University of Michigan, Ann Arbor, Mich., USA) for helpful advice, and to Mrs. Yasuyo Miyahara (Kochi Medical School) for her technical assistance with the measurement of MLCK activity. We thank Ms Eva Garcia del Saz (Kochi Medical School) for her careful reading of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Odani, K., Kobayashi, T., Ogawa, Y. et al. ML-7 inhibits exocytosis of superoxide-producing intracellular compartments in human neutrophils stimulated with phorbol myristate acetate in a myosin light chain kinase-independent manner. Histochem Cell Biol 119, 363–370 (2003). https://doi.org/10.1007/s00418-003-0531-6
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00418-003-0531-6