Regulation of permeabilized endothelial cell retraction by myosin phosphorylation.

Regulation of permeabilized by myosin phosphorylation

endothelial

cell retraction

ROBERT B. WYSOLMERSKI AND DAVID LAGUNOFF Department of Pathology, St. Louis University School of Medicine,

WYSOLMERSKI, ROBERT B., AND DAVID LAGUNOFF~~~Ulation of permeabilixed endothelial cell retraction by myosin phosphorylation. Am. J. Physiol. 261 (Cell Physiol. 30): C32C40, 1991.-Permeabilized endothelial cell monolayers retracted on exposure to ATP and Ca2+. ADP, inosine triphosphate (ITP), GTP, adenosine 5’-(y-thio)triphosphate (ATP$S), and 5’-adenylylimidodiphosphate failed to support retraction. However, ATP$S, a substrate for myosin light-chain kinase (MLCK) but not myosin adenosinetriphosphatase (ATPase), combined with ITP, a substrate for myosin ATPase but. not MLCK, supported retraction. Two MLCK pseudosubstrate peptides, M5 and SM-1, inhibited endothelial cell retraction equally and more effectively than myosin kinase-inhibitory peptide with a sequence based on the phosphorylated site of myosin light chain. M5 was shown to inhibit thiophosphorylation of endothelial cell myosin light chains. Endothelial cells incubated with exogenous unregulated kinase in the presence of ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid retracted on addition of ATP. This retraction was accompanied by thiophosphorylation of the 19 kDa myosin light chains in the presence of ATPr”S. The N-ethylmaleimide-modified subfragment 1 of myosin heads, a specific inhibitor of actin-myosin interaction, prevented retraction. These data add support to the proposal of a central role for MLCK activation of myosin in endothelial retraction. myosin light-chain kinase; calcium calmodulin-independent myosin light-chain kinase; M5; SM-1; N-ethylmaleimide-modified subfragment 1; edema; actin; contractile proteins

SYSTEM is lined by an intact monolayer of endothelial cells (ECs) that form the structural and functional barrier to fluid and solute exchange. Disruption of this intimal lining results in failure of the blood vessel’s impermeable barrier and thus the development of edema. A range of agents that increase microcirculatory permeability (21, 35) cause cultured ECs to retract (27,32). Two possibilities could account for these events: changes in the interendothelial cell junctions and/or activation of the intrinsic contractile activity of the ECs. It is the latter hypothesis that we have pursued. Actin and myosin are believed to mediate a variety of contractile events in nonmuscle cells, including ECs (2, 28, 34). We have used the phosphorylation hypothesis for regulation of smooth muscle contraction as a model for EC retraction. It is generally accepted that phosphorylation of the 20,000-Da light chain of myosin is required to activate the myosin adenosinetriphosphatase (ATPase) essential for contraction. This process is catalyzed by myosin light-chain kinase (MLCK), a Ca”+-

St. Louis, Missouri

63104

calmodulin-dependent enzyme responsible for transfer of y-phosphate from ATP to the myosin light chain. Functionally skinned muscle preparations have been extensively utilized to study the relationship between phosphorylation and tension development under controlled conditions. The virtue of this-preparation is the capability provided of complete control of solutions bathing the contractile proteins. In recent years, several techniques have become available to similarly render nonmuscle cells permeable to otherwise impermeant molecules. Akin to skinned preparations, the use of permeabilized cells has contributed substantially to understanding the mechanism of nonmuscle cell contraction. We have previously shown that retraction is dependent on an adequate supply of cell ATP and Ca2+ and is closely associated with changes in the actin network within the cell (32, 33). To further pursue the molecular basis of EC retraction, we developed a permeabilized EC system that exhibits a retractile response when exposed to ATP and Ca2+ (34). The permeabilized preparation exhibited thiophosphorylation of myosin light chains, which was concomitant with retraction, and both the thiophosphorylation and retraction were prevented bY removal of MLCK and restored by replacement of purified MLCK together with Ca2+ and calmodulin. In this report, we present experiments with the permeabilized EC preparation that add support to our contention that EC utilize a smooth musclelike myosin-based contractile system for retraction.

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$1.50 Copyright

MATERIALS

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METHODS

Cell culture. The bovine pulmonary artery EC line established by Del Vecchio and Smith (10) was obtained from American Type Culture Collection. Cells were maintained in Eagle’s minimal essential medium supplemented with 1 mM glutamine, 10% fetal calf serum, 50 U/ml penicillin, and 50 pg/ml streptomycin. Cells were maintained at 37°C in a humidified 5% COZ-95% air atmosphere. Cells used in these studies were 7 days postconfluent. Permeabilized monolayers. EC monolayers were washed with Dulbecco’s phosphate-buffered saline (DPBS), pH 7.2, and flooded with 2 ml of buffer A [20 mM piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES), 10 mM imidazole, 50 mM KCl, 1 mM ethylene glycol-bis(P-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA), 1 mM MgSO,, 0.2 mM dithiothreitol (DTT), 5 pg/ml aprotinin, 5 pg/ml leupeptin, 10 pg/ml

0 1991 the American

Physiological

Society

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (157.089.065.129) on October 23, 2018. Copyright © 1991 American Physiological Society. All rights reserved.

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soybean trypsin inhibitor, 0.5 mM benzamidine, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF), pH 6.51 containing varying concentrations of saponin for 10 min at 37°C. EC permeabilization was monitored microscopically with 0.1% trypan blue and rhodamine-phalloidin. Biochemical measurements of cytosolic ATP (33) lactate dehydrogenase (LDH; 20) and ,&hexosaminidase (20) released into the extracellular medium were performed. Maximal release of ATP, LDH, and ,&hexosaminidase was determined by flooding cultures with 2 ml of buffer A containing 0.5% Triton X-100 for 10 min at 37°C. The binding of rhodamine-phalloidin to EC F-actin was also used as a measure of permeabilization. For this assay, cultures were fixed and stained and the rhodaminephalloidin extracted as outlined previously (33). Permeabilized monolayers were washed with 2 ml of buffer A without saponin before addition of stimuli in buffer B (50 mM KCl, 25 mM PIPES, 2 mM MgS04, 1 mM EGTA, 0.2 mM DTT, 5 pg/ml aprotinin, 5 pg/ml leupeptin, 5 pg/ml soybean trypsin inhibitor, 0.5 mM benzamidine, 0.1 mM PMSF, pH 7.0) for the desired intervals. The free Ca”+ concentrations of buffer B, a Ca”‘-EGTA-buffer system, were determined by the methods of Bers (3). Quantitation of EC retraction. Stimulation medium was removed at the desired times, and cultures were fixed with freshly prepared 3% formaldehyde in buffer A for 30 min. Fixed cultures were stained with rhodaminephalloidin for visualization of F-actin as outlined previously (32). EC retraction was documented by two methods. For both techniques, random micrographs of control and stimulated monolayers were taken at x500, and the negatives were enlarged ~8. In the first method, intercellular gaps on prints were outlined and digitized for gap area. In the second method, total intercellular gap area was estimated by point counting (31). The two methods gave comparable results. The extent of retraction was determined from the calculated gap area on photomicrographs and expressed as a percentage of the total monolayer area. Total coverage of the culture dish defined no EC retraction. Conditions under which experiments were performed did not allow for 100% retraction (cell detachment). Purification of proteins. Human platelet myosin was purified from outdated platelets obtained from the American Red Cross, Missouri Affiliate, following the methods of Sellers et al. (29). Polyclonal antibodies to purified human platelet myosin were raised in New Zealand White rabbits. An immunoglobulin (Ig) G fraction was purified from pooled rabbit serum as follows. Ammonium sulfate was added to 25% saturation and serum centrifuged at 26,000 g for 20 min. The supernatant was brought to 55% saturation with ammonium sulfate and centrifuged at 26,000 g for 20 min. The pellet was dissolved in 10 ml of DPBS containing 0.1% sodium azide (DPBS-NaN3) and dialyzed for 24 h against 6 liters of DPBS-NaN3. The IgG fractions were aliquoted and stored at -8OOC. The polyclonal IgG fraction (15 mg/ml) used for immunoprecipitation studies recognizes human platelet

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myosin heavy chains as well as myosin heavy chains prepared from bovine pulmonary artery and human umbilical vein EC. Smooth muscle myosin was prepared from bovine uteri as outlined by Adelstein and Klee (1). Myosin subfragment 1 (SJ was isolated following the procedure of Weeds and Taylor (30). For preparation of N-ethylmaleimide-modified-&, (NEM-S1), the protocol of Cande (6) was followed. NEM-S1 was stored in 50% glycerol at -2OOC until used. Smooth muscle MLCK was prepared from chicken gizzards following a modification (34) of the procedures outlined by Adelstein and Klee (1). The specific activity of the purified smooth muscle MLCK was 25 prnol. mg protein-l. min-l when assayed with mixed light chains isolated from turkey gizzards. Unregulated MLCK was prepared by tryptic digestion of MLCK in the presence of bound calmodulin as described by Adelstein et al. (2). Thiophosphorylation. Permeabilized monolayers were incubated with 100 PC1 adenosine 5’-y-[35S]thiotriphosphate (ATPr”S) in buffer B under varying experimental conditions for 10 min at 37°C. Cultures were washed twice with DPBS, pH 7.3, scraped into 50 ~1 of Laemmli sample buffer (19), and electrophoresed on 7.5-12% gradient polyacrylamide gels. Immunoprecipitation of thiophosphorylated EC myosin was performed as described previously (34). Synthetic peptides. The synthetic peptide inhibitor M5 (5, 18) was generously provided by Dr. Edwin Krebs, University of Washington. M5 has the following sequen ce: Lys-Arg-Arg-Trp-Lys-Lys-Asn-Phe-Ile-AlaVal-Ser-Ala-Ala-Asn-Arg-Phe-Gly-NH2. The first 17 amino acids are identical to those found in position 577593 of rabbit skeletal muscle MLCK. SM-1 was synthesized according to the amino acid sequence of residues 480-501 of smooth muscle MLCK by the Saint Louis University Peptide Center employing a stepwise solidphase synthesis utilizing a Du Pont RAMPS peptide synthesizer. The N-tert-butoxy-carbonyl protection methodology was used, and final cleavage was with anhydrous HF. SM-1 was purified following the procedure detailed by Kemp et al. (17). Peptide purity and concentration were assessedby quantitated amino acid analysis. SM-1 has the following sequence: Ala-Lys-Lys-Leu-SerLys-Asp-Arg-Met-Lys-Lys-Tyr-Met-Ala-Arg-ArgLys-Trp-Gln-Lys-Thr-Gly. Myosin kinase-inhibitory peptide (MKIP; 23) was purchased from Peninsula Laboratories (Belmont, CA; MKIP = Lys-Lys-Arg-Ala-AlaArg-Ala-Thr-Ser-NH2). Polyacrylamide gel electrophoresis. Gel electrophoresis was performed in 7.5-12% gradient vertical slab gels using the buffer system of Laemmli (19). Gels were stained with Coomassie blue, destained, incubated in EN3HANCE, dried at 60°C and exposed to Kodak XOMAT X-ray film. For estimation of molecular mass, Pharmacia low-molecular-weight calibration standards were used as follows: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and X-lactalbumin (14.4 kDa).


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Cell permeabilization. We developed a saponin-permeabilized EC preparation to serve as a functional model of the intact EC. Experiments were performed with monolayers incubated in buffer A to determine the minimum concentration of saponin necessary to permeabilize ECs. Permeabilization was monitored by measuring the release of cellular ATP, LDH, and P-hexosaminidase. Figure 1 depicts the response to increasing concentrations of saponin. Loss of cytosolic ATP was detectable at 5 @g/ml saponin, reaching a maximum at 20 yg/ml, whereas the threshold for LDH release was 20 pg/ml saponin. No significant release of /I-hexosaminidase, a lysosomal marker, occurred at saponin concentrations ~50 pug/ml. Maximal release of cellular ATP, LDH, and /3-hexosaminidase occurred at 100 /*g/ml saponin. To further evaluate permeabilization, the binding of rhodamine-phalloidin to F-actin was determined. A saturating concentration of rhodamine-phalloidin was included in buffer A during permeabilization. After a lomin incubation, cultures were washed thoroughly with DPBS and the rhodamine-phalloidin bound to the monolayer extracted and quantitated as described previously (33). The threshold for rhodamine-phalloidin binding to F-actin occurred at 15 pg/ml saponin. Microscopic examination revealed that treatment with 15 pg/ml saponin permeabilized only 60% of the monolayer, whereas treatment with 25 pg/ml saponin rendered 100% of the cells permeable. At 25 pg/ml, the concentration chosen for the experiments, saponin induced the loss of 94 + 3% of the cytosolic ATP and 41 f 5% LDH, whereas /?-hexosaminidase release was only 4 + 3% above that which occurred in controls. At this concentration of saponin, 72 f 6% of maximal binding of phalloidin to actin was achieved. 100

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Monolayers treated with 25 pug/ml saponin (Fig. 2) exhibit a complex pattern of actin filaments closely resembling that of intact cells (32). Stress fibers and the complex arrangement of paranuclear filamentous actin were little affected by permeabilization. The dense peripheral band (DPB) of actin was present but had lost some definition. These observations show that treatment of EC monolayers with 25 pg/ml saponin produced a selective permeabilization of the plasma membrane without significantly altering the EC actin filament distribution. Ca2+- and ATP-dependent retraction of permeabilized ECs. Rhodamine-phalloidin-stained control monolayers

exhibited a cohesive sheet of polygonal cells that covered the substratum. A DPB of F-actin was present at the cell margins that clearly delineated each cell. When permeabilized monolayers were exposed to both 100 yM free Ca2+ and 250 PM ATP, extensive cell retraction occurs (Fig. 3). The DPB of actin and actin stress fibers seemed to contract toward the center of the cell exposing substratum, leaving a limited number of actin strands ra-

FIG. 2. Fluorescence micrograph of F-actin distribution in confluent EC monolayer permeabilized with 25 pg/ml saponin. Control cultures were incubated in 250 FM Na-ATP, 1 mM EGTA for 10 min in buffer B. No retraction occurs in absence of Ca’+. Only occasional gap (triangle) is present within monolayer. x400.

Fg/ml

FIG. 1. Effect of saponin concentration on release of cytosolic ATP lactic dehydrogenase (LDH), &hexosaminidase, and binding of rhodamine-phalloidin to F-actin. Medium was assayed for release of LDH and P-hexosaminidase. ATP content and binding of rhodamine-phalloidin were determined on endothelial cell (EC) extracts as described previously (35). Each point is mean -+ SD of 5 experiments. ATP (circles), LDH (diamonds), P-hexosaminidase (triangles), bound rhodamine-phalloidin (squares).

FIG. 3. Permeabilized monolayers exposed to 250 FM Na-ATP, 100 PM free Ca” in buffer B for 10 min. Severe EC retraction occurs within 10 min. Cells do not detach from substratum. Actin filaments appear to circumferentially retract toward nucleus. x400.


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diating outward. The Ca”+- and ATP-dependent retraction caused an extensive rounding and bulging of individual cells. At the concentrations of ATP and free Ca2+ used, uniform retraction was elicited. Cultures exposed to either Ca2+ or ATP alone showed no alterations in the F-actin distribution, and no detachment of cells from the dish was observed. Figure 4 illustrates the effect of altering the free Ca”’ and ATP concentration on the extent of EC retraction. All experiments were performed in buffer B for 10 min at 37OC. The monolayers were fixed and stained with rhodamine-phalloidin, and retraction was assessedmorphometrically. When permeabilized monolayers were incubated with increasing concentrations of Ca”+ and ATP, a graded increase in the extent of retraction occurred. Monolayers exposed to 1 mM Na-ATP and 0.5 mM free Ca2’ exhibited maximal retraction. At free Ca2+ concentrations between 0 and 500 PM and ATP concentrations between 0 and 1 mM concentrations, the permeabilized cultures re tracted to varying degrees with the extent of retraction being dependent on the free Ca2+ and Na- ATP concentrations. Having demonstrated a differential retractile response to varying Ca”’ concentrations, we sought to determine the sensitivity of permeabilized EC myosin light-chain phosphorylation to free Ca2’. Permeabilized cultures were incubated in ATPT”~S in Ca2+-EGTA buffers for 10 min at 37°C. Figure 5, A and B, shows increasing myosin light chain phosphorylation in response to varying free Ca2+ concentrations ranging from low6 M to 5 X 10B4 M. Maximal thiophosphorylation occurred at 50 FM free Ca2+. No thiophosphorylation of the 19 kDa myosin light chain occurred in the presence of 1 mM EGTA (Lane 1). Nucleotide requirement for EC retraction. The addition of increasing concentrations of ATP to permeabilized monolayers in the presence of 100 PM free Ca2’ caused a graded retraction of cells (Fig. 4). Permeabilized cultures incubated with 100 PM free Ca2+, 250 PM Na-ATP for 10 min exposed 24 t 6% of the substratum, whereas controls treated with either ATP or Ca2’ alone exposed only 3-4%. The ability of other nucleotides to support EC retraction was negligible. Monolayers incubated in the presence of 1 mM ADP, GTP, cytidine triphosphate,

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40 zi 5 30 A

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500 Na/ATP ,uM

FIG. 4. Effects of Na-ATP and free Ca2’ on EC retraction. Monolayers were permeabilized with 25 pg/ml saponin and then incubated in buffer B containing differing concentrations of Na-ATP and Ca” for 10 min at 37OC, fixed, and stained with rhodamine-phalloidin. Retraction was quantitated as outlined in MATERIALS AND METHODS. Each point is mean rf_: SD of 5 experiments.

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inosine triphosphate (ITP), adenosine 5’-(y-thio)triphosphate (ATP$S) or 5’-adenylylimidodiphosphate (AMP-PNP) showed no significant retraction. To further probe the mechanism of endothelial cell retraction, we utilized ITP and an ATP analogue, ATPyS. ATPyS can be utilized as a substrate by MLCK to thiophosphorylate myosin light chains (8, 26) but cannot be used as a substrate for myosin ATPase activity (8, 26). In contrast, ITP supports myosin ATPase activity but is not a substrate for MLCK (22, 26). Figure 6 illustrates the effects of ATPyS and ITP on permeabilized endothelial cell retraction. Cultures incubated in the presence of 100 PM ATPyS (Fig. 7A) or ITP (Fig. 7B) for 10 min in buffer B exhibited minimal retraction and only minor changes in the actin cytoskeleton. In contrast, permeabilized monolayers incubated first in 100 PM ATP+, 100 PM free Ca”+ for 10 min, and then in 100 FM ITP retracted rapidly (Fig. 7C), resulting in exposure of 52% of the culture dish (Fig. 6). Actin filaments retracted toward the center of the cell and exhibited a prominent circumferential pattern. Extensive rounding of individual cells occurred. Small fragments of F-actin remained adherent to the substratum. The rate of retraction on addition of ITP after preactivation with ATPyS was approximately twice that of ATP controls, whereas the extent of retraction was only minimally altered (Fig. 6). We next utilized unregulated MLCK to test the role of MLCK in endothelial cell retraction. When purified MLCK is proteolytically cleaved under controlled conditions, it retains its kinase activity in the absence of Ca2+. When added to permeabilized cultures in the presence of 2 mM EGTA, 100 PM Na-ATP, conditions that do not permit retraction by permeabilized cells, addition of unregulated kinase supported retraction (Fig. 8). We then sought to determine whether retraction in the presence of unregulated kinase and the absence of Ca”’ was accompanied by phosphorylation of myosin light chains. Figure 9A shows the thiophosphorylation pattern from whole permeabilized cells, whereas Fig. 9B shows analysis of immunoprecipitated EC myosin. Incubation of monolayers with ATPy”“S, 2 mM EGTA resulted in minimal thiophosphorylation of myosin light chains [Fig. 9, A (lane 1) and B (lane I)]. In contrast, when unregulated kinase was added under the same conditions, thiophosphorylation of myosin light chains occurred [Fig. 9, A (lane 2) and B (lane Z)]. Synthetic peptide inhibitors. We tested the effects of three synthetic peptide inhibitors of MLCK, M5, SM1, and MKIP, on EC retraction. Permeabilized monolayers were preincubated in buffer C (154 mM NaCl, 5.6 mM KCl, 10 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, 1 mM MgC12, 2 mM EGTA), pH 7.3, containing different concentrations of peptide inhibitors. After a 20-min incubation, cultures were flooded with buffer C containing 100 PM ATP to initiate retraction. Cultures were incubated at 37°C for 10 min, fixed, stained, and retraction quantitated as outlined in MATERIALS AND METHODS. All three peptides inhibited retraction in a dose-dependent manner (Fig. 10). M5 and SM-1 were equally effective inhibitors at 25 ,uM, inhibiting retraction 57 and 52%, respectively. In contrast,


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FIG. 5. A: autoradiogram of representative experiment of immunoprecipitated thiophosphorylated myosin light chains from permeabilized EC monolayers. Lanes 2-7 represent thiophosphorylation of myosin light chains in presence of varying free Ca*+ concentrations. Lane 1: 1 mM EGTA; lane 2: 500 PM free Ca*+; lane 3: 250 PM free Ca*+; lane 4: 100 KM free Ca*+; lane 5: 50 PM free Ca’+; lane 6: 25 ,uM free Ca’+; lane 7: 1 bM free Ca’+. B: dependence of myosin light-chain (MLC) thiophosphorylation on free Ca”. Increase in phosphorylation was confirmed by comparing densitometric scans of Coomassie bluestained myosin heavy chains (MHC) to thiophosphorylated light chains. Ratio of relative densitometric units from the autoradiogram and Coomassie bluestained gels were determined (MLC/ MHC). Ratio corrects for differences in protein loading and allows relative increase in myosin light-chain phosphorylation to be determined.

NEM-S1 inhibition. NEM-S1 as well as NEM-modified heavy meromyosin bind to F-actin and block the binding of native myosin (6), thus preventing production of force necessary for actomyosin contraction. Permeabilized monolayers were preincubated in differing concentrations of NEM-S1 for 15 min before the addition of buffer B containing 100 PM Na-ATP, 100 PM free Ca2+, and NEM-S1 (Table 1). NEM-S1 at 2 mg/ml completely inhibited EC retraction. Unmodified S1 had no effect on EC retraction. DISCUSSION

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FIG. 6. Effects of adenosine 5’-(y-thio)triphosphate (ATP$S) and ITP on permeabilized EC monolayers. All incubations were carried out in buffer B containing 100 PM free Ca”+. Monolayers treated with either ATPrS (up triangles) or ITP (down triangles) alone retract minimally when compared with ATP controls (circles). In contrast, monolayers preincubated with ATP$S, 100 FM free Ca’+, and subsequently incubated with 100 PM ITP (squares) retract at approximately twice the rate of ATP controls, whereas extent of retraction was only minimally altered.

MKIP required a five times greater concentration to achieve comparable inhibition. When monolayers treated with 50 PM M5 peptide were incubated with 100 /IM ATP for as long as 15 min, no retraction was evident (Fig. 11). In parallel with the inhibitor studies, we investigated thiophosphorylation of myosin light chains. Figure 12 is an autoradiogram of immunoprecipitated thiophosphorylated myosin light chains from permeabilized EC treated with M5. Cultures incubated with 100 yCi ATPT~~S, 100 PM free Ca’+, and increasing concentrations of M5 (lanes 2-4) showed a marked decrease in the extent of myosin light-chain thiophosphorylation.

We have previously described the utility of a permeabilized EC system for the investigation of the role of MLCK in EC retraction (34). The benefit of having an intact contractile system that can be modified in a variety of ways without regard for the plasma membrane is obvious and has been effectively used in the study of smooth muscle (9, 15, 16) and nonmuscle cells (7, 11, 34). The disadvantages of permeabilization are the severe perturbation of the plasma membrane and possible interactions between it and the contractile system. In the permeabilized EC system, although the cells remain in their original spread form, they do lose the ability to respond to agonist stimulation. This loss limits but does not obviate the use of the permeabilized cell system. The experiments on the permeabilized ECs we have carried out are based on the hypothesis that the endothelial contractile system is analogous to the system in smooth muscle cells. The first approach we used to test the correspondence of the EC retractile mechanism to that of the smooth muscle cell was to examine its nucleotide specificity. As is the case for smooth muscle contraction, only ATP was able to support retraction. The ability of MLCK to phosphorylate myosin light chains is highly specific for ATP and not supported by other nucleotides (25). However, MLCK is able to use ATPrS to activate myosin ATPase, via thiophosphorylation of myosin light chains (8, 9), but ATPrS is not a


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FIG. 8. F-actin distribution in monolayers incubated with unregulated myosin light-chain kinase (MLCK). Cultures were permeabilized and incubated in buffer B containing 2 mM EGTA, 50 pg/ml unregulated MLCK, and 100 PM Na-ATP. Retraction is supported by kinase in presence of EGTA. Severe EC retraction results and F-actin filaments contracted down around nucleus. x400.

FIG. 7. A and B: fluorescence micrographs of F-actin distribution in permeabilized monolayers exposed to either 100 PM ATPrS (A) or 100 ,nM ITP (B) in buffer B containing 100 PM free Ca’+. After lo-min exposure only minimal endothelial retraction results. Occasional gap develops between adjacent cells, and no cell rounding or detachment from substratum occurred. Some loss of dense peripheral band occurs on exposure to either ATPrS or ITP. x400. C: permeabilized monolayer preincubated in 100 PM ATP+, 100 FM free Ca”+ in buffer B for 10 min and then exposed to 100 PM ITP. Incubation with both nucleotides supports dramatic cell retraction. F-actin retracts centrally, displaying prominent circumferential pattern. Extensive cell rounding occurs so that cells bulge out of plane of focus. No cell detachment from culture dish was noted. x400.

substrate for myosin ATPase. On the other hand, ITP is a functional substrate for myosin ATPase but not for MLCK (9, 22). Neither of these nucleotides by themselves support retraction; however, in concert the two should allow retraction. This prediction was fulfilled in

FIG. 9. A: autoradiogram of thiophosphorylated proteins from permeabilized EC monolayer. Lane 1: monolayer incubated in presence of 100 PCi of ATP-y?S, 2 mM EGTA for 10 min. No thiophosphorylation of myosin light chains occurs in presence of EGTA. Lane 2: permeabilized cultures incubated in presence of 2 mM EGTA, 50 pg/ml unregulated MLCK, and 100 KCi of ATPr”S result in thiophosphorylation of 19 kDa myosin light chains. Lane 3: EC monolayer incubated results in thiophosphoin presence of 100 PM Ca”+, 100 j&i ATPr% rylation of endogenous EC myosin light chains. B: immunoprecipitation studies were performed to establish that 19 kDa thiophosphorylated protein from permeabilized extracts was myosin light chain. In presence of 2 mM EGTA, 100 PCi ATPr’S minimal thiophosphorylation of myosin light chain occurred (lane 1). Monolayers incubated in presence of 2 mM EGTA, 100 &i ATPr%, and 50 Kg/ml unregulated kinase exhibited strong thiophosphorylation of endogenous EC myosin light chains (lane 2).

the permeabilized system indicating not only that MLCK activity is required but that an additional step, with the nucleotide specificity of myosin ATPase, is essential for retraction. Studies with skinned smooth muscle preparations have shown that incubation with ATPyS leads to phosphorylation of myosin light chains and that sub-


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10. Inhibition of Ca*+ and ATP induced EC retraction by M5, SM-1, and myosin kinase-inhibitory peptide (MKIP). EC monolayers were permeabilized with 25 fig/ml saponin and preincubated for 20 min with various peptide concentrations and fixed 10 min after being exposed to ATP and Ca*+. Extent of retraction was assessed as outlined under MATERIALS AND METHODS. EC retraction was compared with permeabilized monolayers incubated without peptides. Each point is mean rf~ SD of 3 experiments. M5 (circles), SM-1 (triangles), MKIP (squares).

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FIG. 12. Autoradiogram exhibiting M5 inhibition of thiophosphorylation. Permeabilized monolayers were preincubated with various peptide concentrations for 20 min. Pretreated monolayers were then exposed to 100 PM free Ca*+, 100 &i ATPy9 for 10 min. Lane 1: control monolayer exposed to 100 FM free Ca’+, 100 &i ATPr% only; lane 2: permeabilized monolayer preincubated with 1 PM M5; lane 3: 25 PM M5; lane 4: 50 FM M5.

TABLE

1. Effect of NEM-S1

on EC retraction

Conditions

Control Buffer B, Experimental Buffer B Buffer B Buffer B

Ca’+, ATP conditions + 2 mg/ml S1 Ca’+, ATP + 500 mg/ml NEM-SI, Ca’+, ATP + 2 mg/ml NEM-S1, Ca*+, ATP

Extent Retraction,

of %

47+-3.5 46+6 31+6 6+2

Endothelial cell (EC) monolayers were permeabilized with 25 pg/ml saponin in buffer A for 10 min at 37°C. Cultures were preincubated in buffer B alone, buffer B containing unmodified Si heads (S1), or buffer B containing NEM-S, for 20 min before addition of ATP and Ca’+. Monolayers were incubated for an additional 10 min in buffer B containing 100 rM free Ca*+ 100 PM ATP. EC retraction was determinedas outlinedin MATERIALS AND METHODS. FIG. 11. Fluorescence micrograph of F-actin abilized EC monolayer preincubated with 50 FM min, and then exposed to 100 PM ATP, 100 additional 10 min. M5 inhibited ATP-induced gaps are present within monolayer, but there is tion or loss of F-actin. x400.

distribution in permeM5 in buffer C for 20 PM free Ca” for an EC retraction. Small no evidence of retrac-

sequent addition of ATP results in tension development. Our results obtained with ATPyS and ITP are in accord with the previous studies performed in permeabilized muscle (8, 9) and nonmuscle (26) cell preparations. Our next approach was to examine the effects of synthetic peptides known to inhibit MLCK phosphorylation of myosin. In recent years a self-regulatory mechanism for MLCK activation has been invoked based on the concept of autoinhibition (18) by a pseudosubstrate domain (12, 24). This model focuses on clusters of basic

amino acids, believed to be inhibitory sequences, that reside on the kinase molecule. These inhibitory sequences are similar to sequences of the substrate and capable of blocking the catalytic site of the kinase in the inactive form. Activation alters the interaction between the active site and the pseudosubstrate domain, thereby removing the inhibition and allowing the kinase to interact with substrate. Our studies were performed with peptide analogues based on 1) M5, a portion of the regulatory domain of skeletal muscle MLCK (residues 577-593); 2) SM-1, a pseudosubstrate inhibitory site of smooth muscle MLCK (residues 480--501), and 3) MKIP, a peptide analogue based on the sequence around serine-19 of myosin light chain, the serine phosphorylated by MLCK (residues 11-19). All three peptides inhibited retraction of permeabilized cells in a dose-


MYOSIN

LIGHT-CHAIN

KINASE

AND

dependent manner. Both M5 and SM-1 inhibited EC retraction in a dose-dependent manner (Fig. 10) over a range of concentrations similar to that reported for SM1 and R20 in skinned smooth muscle preparations (16). M5, the only peptide so tested, inhibited myosin lightchain phosphorylation in a dose-dependent manner. An important caveat with respect to the MLCK inhibiting peptides is that they also bind Ca2’-calmodulin (4, 17, 18) and it is conceivable that they inhibit retraction by blocking Ca2+- calmodulin activation of MLCK or another Ca2+-calmodulin-activated enzyme rather than by blocking the active site of MLCK. Blumenthal et al. (4) have shown that M5 in vitro does in fact inhibit several Ca2+-calmodulin-dependent enzymes other than MLCK. Given the limitation of the peptide studies and the requirement for the inclusion of Ca2+-calmodulin in the previously reported MLCK extraction-reconstitution experiments, an experiment was designed to test the effect of myosin light-chain phosphorylation in the absence of Ca”+-calmodulin using unregulated MLCK. Controlled proteolysis of MLCK removes the carboxyl terminal regulatory portion of the molecule, making the modified kinase Ca”+-calmodulin independent. Addition of unregulated kinase to permeabilized cells induced retraction in the presence of 2 mM EGTA with no Ca2+ added. These results are in agreement with several studies in intact (13, 14) as well as skinned smooth muscle (2, 16) and nonmuscle preparations (7). In the final set of experiments we sought to directly test the involvement of myosin in retraction. NEM-S1 heads bind irreversibly to F-actin, blocking the association of native myosin with actin and thereby preventing myosin from exerting a contractile force. The ability of NEM-S1 heads to prevent retraction of permeabilized EC provided evidence that myosin is essential for the retractive phenomenon. The present experiments extend the evidence from the previously reported reconstitution experiments for asserting that a myosin-based contractile system is active in ECs. The retractile events this system is capable of sustaining appear to be sufficient to explain the retraction induced by a range of agonists in ECs with intact plasma membranes, events we and others have proposed underlie the increase in endothelial permeability to macromolecules caused by those agonists (21, 35). Direct evidence for this conclusion from experiments with intact cells is still scant, but the knowledge acquired on the contractile system of the EC now makes it possible to begin development of direct tests of the hypothesis that an actin-myosin contractile mechanism is essential for increased vascular permeability in vivo. This work was supported in part by a Biomedical Research Grant RR-05388 to St. Louis University (to R. B. Wysolmerski) and by National Heart, Lung, and Blood Institute Grants HL-45788 (to R. B. Wysolmerski) and HL-30572 (to D. Lagunoff). Address reprint requests to R. B. Wysolmerski. Received

7 December

1990; accepted

in final

form

15 February

1991.

REFERENCES 1. ADELSTEIN, R. S., AND tion of smooth muscle

C. B. KLEE. Purification and characterizamyosin light chain kinase. J. BioZ. Chem.

ENDOTHELIAL

CELL

RETRACTION

c39

256: 7501-7509, 1981. 2. ADELSTEIN, R. S., M. D. PATO, J. R. SELLERS, P. DE LANEROLLE, AND M. A. CONTI. Regulation of actin-myosin interaction by reversible phosphorylation of myosin and myosin kinase. Cold Spring Harbor Symp. Quant. Biol. 146: 921-928, 1981. 3. BERS, D. M. A simple method for the accurate determination of free [Cal in Ca-EGTA solutions. Am. J. Physiol. 242 (CeZZ PhysioZ. 11): C404-C408, 1982. 4. BLUMENTHAL, D. K., H. CHARBONNEAU, A. M. EDELMAN, T. R. HINDS, G. B. ROSENBERG, D. R. STORM, F. F. VINCENZI, J. A. BEAVO, AND E. G. KREBS. Synthetic peptides based on the calmodulin-binding domain of myosin light chain kinase inhibit activation of other calmodulin dependent enzymes. Biochem. Biophys. Res. Commun. 156: 860-865, 1988. 5. BLUMENTHAL, D. K., AND E. G. KREBS. Preparation and properties of the calmodulin-binding domain of skeletal muscle myosin light chain kinase. Methods Enzymol. 139: 115-126, 1987. 6. CANDE, W. Z. A permeabilized cell model for studying cytokinesis using mammalian tissue culture cells. J. CeZZ BioZ. 87: 326-335, 1980. 7. CANDE, W. Z., AND R. M. EZZELL. Evidence for regulation of lamellipodial and tail contraction of glycerinated chicken embryonic fibroblasts by myosin light chain kinase. CeZZ Motil. Cytoskeleton 6: 640-648, 1986. 8. CASSIDY, P., P. E. HOAR, AND W. G. L. KERRICK. Irreversible thiophosphorylation and activation of tension in functionally skinned rabbit ileum strips by [““S]ATPyS. J. BioZ. Chem. 254: 11148-11153, 1979. 9. CASSIDY, P., AND W. G. L. KERRICK. Superprecipitation of gizzard actomyosin and tension in gizzard muscle skinned fibers in the presence of nucleotides other than ATP. Biochim. Biophys. Acta 705: 63-69, 1982. 10. DEL VECCHIO, P. J., AND J. R. SMITH. Expression of angiotensinconverting enzyme activity in cultured pulmonary artery endothelial cell. J. Cell. Physiol. 108: 337-345, 1981. 11. HOLZAPFEL, G., J. WEHLAND, AND K. WEBER. Calcium control of actin-myosin based contraction in Triton models of mouse 3T3 fibroblasts is mediated by the myosin light chain kinase (MLCK)calmodulin complex. Exp. CeZZ. Res. 148: 117-126, 1983. 12. IKEBE, M. Mode of inhibition of smooth muscle myosin light chain kinase by synthetic peptide analogs of the regulatory site. Biochem. Biophys. Res. Commun. 168: 714-720, 1990. 13. ITOH, T., M. IKEBE, G. KARGACIN, D. HARTSHORNE, B. KEMP, AND F. S. FAY. Modulators of myosin light chain kinase activity affect both [Ca”‘] and contraction in single smooth muscle cells. In: Frontiers in Smooth Muscle Research, edited by N. Sperelakis and J. D. Wood. New York: Wiley-Liss, 1990, p. 73-87. 14. ITOH, T., M. IKEBE, G. J. KARGACIN, D. J. HARTSHORNE, B. E. KEMP, AND F. S. FAY. Effects of modulators of myosin light chain kinase activity in single smooth muscle cells. Nature Lond. 338: 164-167, 1989. 15. KARGACIN, G. J., AND F. S. FAY. Physiological and structural properties of saponin-skinned single smooth muscle cells. J. Gen. Physiol. 90: 49-73, 1987. 16. KARGACIN, G. J., M. IKEBE, AND F. S. FAY. Peptide modulators of myosin light chain kinase affect smooth muscle cell contraction. Am. J. PhysioZ. 259 (Cell Physiol. 28): C315-C324, 1990. 17. KEMP, B. E., R. B. PEARSON, V. GUERRIERO, JR., I. C. BAGCHI, AND A. R. MEANS. The calmodulin binding domain of chicken smooth myosin light chain kinase contains a pseudosubstrate sequence. J. Biol. Chem. 262: 2542-2548, 1987. 18. KENNELLY, P. J., A. M. EDELMAN, D. K. BLUMENTHAL, AND E. G. KREBS. Rabbit skeletal muscle myosin light chain kinase. The calmodulin binding domain as a potential active site-directed inhibitory domain. J. BioZ. Chem. 262: 11958-11963, 1987. 19. LAEMMLI, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature Lond. 227: 680-685, 1970. 20. LAGUNOFF, D., AND A. RICKARD. Methods for the study of rat peritoneal mast cell secretion. In: In Vitro Methods for Studying Secretion, edited by A. M. Poisner and J. M. Trefaro. Amsterdam, Netherlands: Elsevier, 1987, p. 13-28. 21. MAJNO, G., AND G. E. PALADE. Studies on inflammation. 1. The effect of histamine and serotonin on vascular permeability: an


c40

22.

23.

24.

25.

26.

27.

28.

MYOSIN

LIGHT-CHAIN

KINASE

AND

electron microscopic study. J. Biophys. Biochem. CytoZ. II: 571585, 1961. MALIK, M. N., M. D. FINKO, AND R. G. HOWARD. Comparison of steady-state kinetics of thiophosphorylated versus unphosphorylated smooth muscle myosin. Arch. Biochem. Biophys. 216: 671684, 1982. PEARSON, R. B., L. Y. MISCONI, AND B. E. KEMP. Smooth muscle myosin kinase requires residues on the COOH-terminal side of the phosphorylation site. J. Biol. Chem. 261: 25-27, 1986. PEARSON, R. B., R. E. H. WETTENHALL, A. R. MEANS, D. J. HARTSHORNE, AND B. E. KEMP. Autoregulation of enzymes by pseudosubstrate prototopes: myosin light chain kinase. Science Wash. DC 241: 970-973,1988. PIRES, E. M. V., AND S. V. PERRY. Purification and properties of myosin light chain kinase from fast skeletal muscle. Biochem. J. 167: 137-146,1977. PORRELLO, K., AND B. BURNSIDE. Regulation of reactivated contraction in teleost retinal cone models by calcium and cyclic adenosine monophosphate. J. Cell BioZ. 98: 2230-2238, 1984. ROTROSEN, D., AND J. I. GALLIN. Histamine type 1 receptor occupancy increases endothelial cytosolic calcium, reduces F-actin, and promotes albumin diffusion across cultured endothelial monolayers. J. CeZZ BioZ. 103: 2379-2387, 1986. SELLERS, J. R., AND R. S. ADELSTEIN. Regulation of contractile

ENDOTHELIAL

CELL

RETRACTION

activity. Orlando,

29.

30.

31.

32.

33. 34.

35.

In: The Enzymes, edited by P. D. Boyer and E. G. Krebs. FL: Academic, 1987, vol. 18, p. 381-395. SELLERS, J. R., M. S. SOBOEIRO, K. FAUST, A. R. BENGUR, AND E. V. HARVEY. Preparation and characterization of heavy meromyosin and subfragment 1 from vertebrate cytoplasmic myosins. Biochemistry 27: 6977-6982, 1988. WEEDS, A., AND R. TAYLOR. Separation of subfragment-l isoenzymes from rabbit skeletal muscle myosin. Nature Lond. 275: 5456, 1975. WILLIAMS, M. A. Quantitative methods in biology. In: Practical Methods in ELection Microscopy, edited by A. M. Glauert. New York: North-Holland, 1977. WYSOLMERSKI, R. B., AND D. LAGUNOFF. The effect of ethchlorvynol on cultured endothelial cells: a model for the study of the mechanism of increased vascular permeability. Am. J. Pathol. 119: 505-512,1985. WYSOLMERSKI, R. B., AND D. LAGUNOFF. Inhibition of endothelial cell retraction by ATP depletion. Am. J. Pathol. 132: 28-37, 1988. WYSOLMERSKI, R. B., AND D. LAGUNOFF. Involvement of myosin light chain kinase in endothelial cell retraction. Proc. NatZ. Acad. Sci. USA 87: 16-20,199O. WYSOLMERSKI, R. B., D. LAGUNOFF, AND T. DAHMS. Ethchlorvynol-induced pulmonary edema in rats: an ultrastructural study. Am. J. PathoZ. 115: 447-457, 1984.


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