Proc. Nati. Acad. Sci. USA Vol. 87, pp. 16-20, January 1990 Medical Sciences Involvement of myosin light-chain kinase in endothelial cell retraction (actin/microfilaments/cytoskeleton/calmodulin) ROBERT B. WYSOLMERSKI* AND DAVID LAGUNOFF Department of Pathology, Saint Louis University School of Medicine, 1402 South Grand Boulevard, Saint Louis, MO 63104 Communicated by Earl P. Benditt, September 26, 1989 ABSTRACT Permeabilized bovine pulmonary artery en- light-chain phosphorylation permits activation of myosin dothelial cell monolayers were used to investigate the mecha- ATPase by actin and is thereby believed to initiate the nism of endothelial cell retraction. Postconfluent endothelial contractile events resulting in the shortening and tension cells permeabilized with saponin retracted upon exposure to development of smooth muscle cells (6-8). ATP and Ca2+. Retraction was accompanied by thiophospho- Skinned preparations of smooth muscle in which the mem- rylation of 19,000-Da myosin light chains when adenosine brane barrier to influx of large molecules and charged small 5'-[y-["SSthioltrlphosphate was included in the dium. Both molecules has been destroyed have been important in ana- retraction and thiophosphorylation of myosin liht chains lyzing the biochemical characteristics of the contractile pro- exhibited a graded quantitative dependence on Ca2+. When cess in these cells (6, 7, 9). Similar preparations offibroblasts permeabilized monolayers were extracted in buffer D contain- (10, 11) and epithelial cells (12, 13) have also been studied. ing 100 mM KCI and 30 mM MgCI2 for 30 min, the cells failed We have developed a preparation of permeabilized ECs that to retract upon exposure to ATP and Ca2+, and no thiophos- has provided us with the opportunity to investigate the role phorylation of myosin light chains occurred. The ability both ofATP, Ca2+, calmodulin, and MLCK in the initiation of EC to retract and to thiophosphorylate myosin light chains was retraction. restored by the addition to the permeabilized, extracted cells of myosin light-chain kinase and calmodulin together but not by EXPERIMENTAL PROCEDURES either alone. These studies indicate that endothelial cell retrac- tion, as does smooth muscle contraction, depends on myosin Cell Culture. The bovine pulmonary artery cell linedeveloped light-chain kinase phosphorylation of myosin light chains. by Del Vecchio and Smith (14) was obtained from American Type Culture Collection (CLL-209). Cells were grown in Ea- gle's minimal essential medium supplemented with 2 mM glu- A major function of the endothelial cell (EC) is to serve as a tamine, 10o or 20%o fetal calf serum, penicillin at 50 units/ml, barrier to fluid and solute flux across the blood vessel wall. and streptomycin at 50 gg/ml. Cells were maintained at 370C in Breakdown ofthis barrier leads to increased permeability and a humidified 5% C02/95% air atmosphere. Cells used in these the development of edema. Since the classic report by Majno studies were 7 days postconfluent (2). and Palade (1) describing opening ofintracellularjunctions on Cell Permeabilization. EC cultures were washed with Dul- exposure to histamine, there has been considerable interest becco's phosphate-buffered saline (DPBS) (pH 7.2) and per- in the mechanism underlying this effect. Two broad possi- meabilized with 2 ml of buffer A (20 mM Pipes/10 mM bilities have been entertained, change in the junctions them- imidazole/50 mM KCI/1 mM EGTA/1 mM MgSO4/0.2 mM selves and intrinsic contractile activity of the ECs. dithiothreitol/5 ,g of aprotonin per ml/5 ug of leupeptin per In situ, lining blood vessels and in postconfluent cultures, ml/10 ,ug of soybean trypsin inhibitor per ml/0.1 mM phe- ECs are flattened polygonal cells without obvious polarity, nylmethylsulfonyl fluoride/0.5 mM benzamidine, pH 6.5) possessing a complex actin distribution with a dense periph- containing 25 ,ug of saponin per ml, incubated at 37°C for 10 eral band, stress fibers, and paranuclear filamentous array min, and washed with 2 ml ofbuffer A without saponin. Care (2). Exposure of ECs in culture to histamine (3, 4), ethchlor- was taken not to allow the permeabilized monolayers to dry. vynol (2, 3), or cytochalasin B (3, 5) induces a reversible Permeabilized monolayers were stimulated to retract on retraction of confluent ECs, leaving gaps between the cells, the addition of 100 ,uM free Ca2' and 100 ,uM exogenous ATP which remain attached to one another by thin processes. in buffer B (50 mM KCI/25 mM Pipes/2 mM MgSO4/1 mM Intracellular ATP and extracellular Ca2' have been shown to EGTA/0.2 mM dithiothreitol/5 ,g of aprotonin per ml/5 ,ug be essential for the retraction induced by these agents, and of leupeptin per mj/10 ,ug of soybean trypsin inhibitor per the retraction is accompanied by extensive changes in actin ml/0.1 mM phenylmethylsulfonyl fluoride/0.5 mM benzami- filament distribution (3). These several observations favor dine, pH 7.0) for 10 min at 370C. Retraction was terminated the contraction hypothesis for intercellular gap formation. by removing the buffer containing ATP and adding 3% ECs, like other eukaryotic nonmuscle cells, contain myo- formaldehyde in buffer A. The free Ca2' concentration of sin, actin, and associated proteins involved in cellular motile buffer B was calculated by the method of Bers (15). activities. For our studies, we have used the control of the Rhodamine Phalloidin Staining of Permeabilized Monolay- contractile apparatus in smooth muscle as a model for EC ers. For actin cytochemistry, cells were grown in Coming contraction. Phosphorylation of the 20-kDa light chains of dishes (35 x 10 mm). Monolayers were permeabilized and the myosin is essential for smooth muscle contraction (6, 7). The cells were stimulated to retract as described above. The phosphorylation is catalyzed by the Ca2", calmodulin- monolayers were fixed in freshly prepared 3% formaldehyde dependent enzyme myosin light-chain kinase (MLCK), in buffer A for 30 min at 220C. Fixed monolayers were washed which transfers the y-phosphate from ATP to myosin. The with buffer A and stained with rhodamine phalloidin (Mo- The publication costs ofthis article were defrayed in part by page charge Abbreviations: EC, endothelial cell; MLCK, myosin light-chain payment. This article must therefore be hereby marked "advertisement" kinase; ATP[y-35S], adenosine-5'-[y-[35S]thio]triphosphate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 16 Downloaded by guest on September 28, 2021 Medical Sciences: Wysolmerski and Lagunoff Proc. Nati. Acad. Sci. USA 87 (1990) 17 lecular Probes) for visualization of F actin as outlined by CaCI2 was added to give a final free concentration of 0.5 mM Barak et al. (16). CaC12. The kinase was then mixed with phenyl-Sepharose Thiophosphorylation. Permeabilized cultures were incu- preequilibrated with 40 mM Tris HCI, pH 7.4/50 mM NaCI/ bated with 100 ACi of adenosine 5'-[y-[35S]thio]triphosphate 0.5 mM CaCl2/1 mM dithiothreitol for 1 hr at 40C. The (ATP[y-35S]) (Amersham) (1 Ci = 37 GBq) in buffer A under phenyl-Sepharose (Sigma) suspension was centrifuged at varying conditions for 10 min at 370C, washed twice with 1000 x g for 10 min, and the soluble MLCK was removed and buffer A and scraped up in 50 pl of SDS sample buffer (17), dialyzed overnight against three 6-liter changes of 20 mM heated for 3 min at 100'C, and electrophoresed as described Tris HCI, pH 7.4/50 mM NaCI/1 mM EGTA/1 mM dithio- below. threitol. The purified kinase was stored at -70'C in 0.1-ml In some instances, myosin was immunoprecipitated fol- aliquots. lowing the method described by Kawamato and Adelstein Assay for MLCK Activity. MLCK activity was determined (18). The washed cultures were flooded with 600 Al of buffer by incubation of 0.1 ml of column fractions with 0.1 ml of 25 C (25 mM Tris'HCl/100 mM sodium pyrophosphate/100 mM mM Tris-HCI, pH 7.3/5 mM MgC12/50 mM KCI/0.2 mM NaF/250 mM NaCI/10 mM EGTA/5 mM EDTA/1% Noni- det CaC12 excess over the EGTA and EDTA present/5 ,g of P-40/0.2 mM phenylmethylsulfonyl fluoride/0.5 mM calmodulin (Biomedical Technologies, Stoughton, MA) per benzamidine/10 ,g of aprotonin per ml/10 ,ug of leupeptin ml/0.5 mg of isolated myosin light chains per ml/Ly-32PJATP per ml/10 ,g of soybean trypsin inhibitor per ml, pH 8.8) sufficient to tube. with a rubber 10 give 300,000 cpm per Column fractions scraped up policeman, made mM with were incubated at 25°C for 10 min and the was respect to disodium sonicated in a bath and assay ATP, sonicator, terminated by addition of 50%o trichloroacetic acid/109o so- incubated on ice for 20 min. The insoluble particulate mate- dium rial was sedimented at 80,000 x g for 10 min in a Beckman pyrophosphate to give a final concentration of 10% TL-100 ultracentrifuge. The soluble EC extracts were incu- trichloroacetic acid and 2% sodium pyrophosphate. Samples bated with an IgG fraction of rabbit antiserum raised against were heated to 90°C for 20 min, cooled on ice and filtered human platelet myosin and generously provided by Robert through Schliecher and Schuell 0.45-,m filters, washed with Adelstein (National Institutes ofHealth), for 2 hr at 4°C prior 5% trichloroacetic acid/1% sodium pyrophosphate, dried, to the addition of prewashed protein A-Sepharose 4B. After and counted in a Beckman LS1801 scintillation counter to an additional 2-hr incubation, the immune complexes bound assess phosphate incorporation into myosin light chains.