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ATP-depleted monolayers, whereas exposure to cyto B resulted in .... Monolayers were stained with Eosin-Coomassie Blue as outlined in Materials and Meth-.
American Journal of Pathology, Vol. 132, No. 1, July 1988 Copyright

American Association of Pathologists

Inhibition of Endothelial Cell Retraction by ATP Depletion

ROBERT B. VYSOLMERSKI, PhD and DAVID LAGUNOFF, MD

From the Department ofPathology, St. Louis University School of Medicine, St. Louis, Missouri

To determine the relationship among endothelial cell (EC) retraction, cell adenosine triphosphate (ATP), and the status ofcellular actin, ATP levels, F-actin content, and cytochemical redistribution in bovine pulmonary artery endothelial cells were assessed. EC monolayers 7 days after confluence were exposed to ethchlorvynol (ECV), histamine, or cytochalasin B (cyto B) for time intervals from 5-90 minutes. All 3 agents resulted in endothelial cell retraction without significant effect on cellular ATP content. Sixty-minute incubation of monolayers in glucose-free media containing antimycin A and 2-deoxyglucose depleted cellular ATP to less than 10% of control levels. ATP

ripheral band of actin to a subcortical position. Cyto B caused loss ofthe dense peripheral band and the longitudinal microfilament bundles. Monolayers depleted of ATP lost their dense peripheral band and exhibited a disorganized, tangled web of microfilaments. Neither histamine nor ECV modified the actin distribution in ATP-depleted monolayers, whereas exposure to cyto B resulted in substantial change in actin with formation of a rim inside the cell membrane and considerable loss of actin filaments. ECV or histamine induced a small reduction in F-actin content while cyto B resulted in a 50% decline in 15 minutes. ATP depletion resulted in a 19% decrease in F-actin, with no further reduction on subsequent exposure to histamine or ECV. Cyto B treatment of ATP-depleted monolayers caused a drop in F-actin content equivalent to that observed in cells with normal ATP levels. These studies indicate that ATP is essential for changes in actin filament distribution and endothelial cell retraction produced by ECV, histamine or cyto B, and make it unlikely that any of these agents acts simply by depolymerization of actin filaments or modification ofthe dense peripheral band, although disruption of the dense peripheral band may facilitate retraction in the presence of adequate levels ofcell ATP. (AmJ Pathol 1988, 132:28-37)

depleted monolayers failed to retract when incubated with ECV, histamine, or cyto B. ATP depletion resulted in loss of the prominent EC margins but only a rare gap between adjacent cells. When ATP levels were allowed to recover, the ability of EC monolayers to retract was restored. Actin filaments in control monolayers localized to a dense peripheral band of actin, a paranuclear complex, and bundles of microfilaments orientated parallel to the long axis ofthe cell. ECV induced complete loss of the dense peripheral band and other changes in the actin disposition. Monolayers exposed to histamine showed a retraction of the dense pe-

cells leaving gaps between the cells. We have extended our studies of the events in endothelial cells (EC) exposed to ECV, histamine or cytochalasin B to an examination ofthe requirement of ATP for intercellular gap formation and the effect of adenosine triphosphate (ATP) depletion on actin filament content and filament distribution in cells exposed to ECV, histamine, and cyto B.

IN RECENT YEARS attention has focused on the integrity of the endothelium and its response to injury. The loss of continuity of the endothelial sheet has been implicated as a primary event in acute edema'3, 4'33 and an early component ofthe development of atherosclerotic lesions.4'5 The mechanisms of endothelial injury are multiple. It is useful to distinguish between nondenuding and denuding injury. In the former the endothelial cells remain in place, lining the vessel but in an altered form, whereas in the latter the cells are sloughed from the vessel wall. We have reported previously6 that in tissue culture, exposure of bovine pulmonary artery endothelial cells (BPAE) or human umbilical vein endothelial cells to ethchlorvynol (ECV) or histamine results in a reversible, nondenuding retraction of confluent endothelial

Supported in part by SCOR: Adult Respiratory Failure HL30572. R.B.W. was supported by a Training Grant HL07050 from the National Institutes of Health. Accepted for publication February 10, 1988. Address reprint requests to David Lagunoff, MD, Department of Pathology, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104. 28

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Materials and Methods Cell Culture The BPAE endothelial cell line developed by Del Vecchio and Smith7 was obtained from American Type Culture Collection (CCL 209). Cells were grown in Eagle's minimal essential medium supplemented with 2 mM glutamine, 10 or 20% fetal calf serum (FCS), 50 U/ml penicillin, and 50 ,ug/ml streptomycin. Cells were cultured in 35 X 10 mm Corning dishes at 37 C in a humidified 5% CO2 atmosphere. Cells used in these studies were 7 days postconfluent. Confluence was defined as complete coverage of the culture surface with uniform polygonal cells with no evident space between cells by phase microscopy at X40.

Cell ATP Determination Cell ATP was determined by the luciferin-luciferase method8 on cell extracts. Control and treated monolayers were rinsed with 2 ml Dulbecco Phosphate Buffered Saline (DPBS), pH 7.3, flooded with 1 ml cold 0.4 M perchloric acid (PCA), scraped up with a rubber policeman, and transferred to a 13 X 100 mm test tube. Culture dishes were washed with an additional 0.5 ml PCA, and the samples combined and incubated on ice for 30 minutes. PCA extracts were centrifuged at 750g, the supernatants removed, and the precipitates allowed to air dry. Immediately before ATP determination the pH of EC extracts was adjusted to 7.0 with KOH. The precipitates were redissolved in 6 N NaOH for protein determination by a modification of the method of Lowry et al.9 For the ATP assay, firefly luciferase was freshly prepared daily by homogenizing 50 mg of desiccated firefly lanterns (Sigma Chemical Co., St. Louis, MO) in 5 ml of cold firefly tail buffer (0.1 M Trizma, 30 mM MgSO4, 3 mM EDTA, 0.5 mM dithiothreitol, pH 7.8). The homogenate was centrifuged at 50,000g for 30 minutes, and the supernatant was removed and diluted 1:10 in buffer and kept on ice until used. A stock solution of D-luciferin, 1 mg/ml (Sigma Chemical Co., St. Louis, MO) was prepared in distilled water and stored on ice protected from light. A standard stock solution of 1 mM disodium ATP (Sigma Chemical Co., St. Louis, MO) was made in distilled water and serially diluted to yield standards containing 1, 2, 5, and 10 picomoles ATP. The final assay medium consisted of 250 ,ul buffer, 5 ,ul of stock luciferin solution, and S or 10 ,ul of ATP standards or samples in PCA. The final solution was vortexed and the luminescent intensity was measured 30 seconds after mixing from a continuous recording of luminescence using an Aminco luminometer and Hewlett-Packard integrator.

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Eosin Coomassie Blue Staining Endothelial cell cultures were washed twice with DPBS, pH 7.3, at 37 C and the agents to be tested then applied to the monolayers. After the desired intervals of exposure, the cultures were washed twice with DPBS and fixed in freshly prepared 2% formaldehyde, 1% glutaraldehyde in DPBS at pH 7.3 for 1 hour at room temperature. The fixed cultures were washed with DPBS and stained with a 60:40 mixture of 0.5% alcoholic acidic eosin Y and 0.125% acidic Coomassie brilliant blue R-250 (final pH 3.5) in methanol:water: glacial acetic acid (5:4:1) for 15 minutes at room temperature. Monolayers were extensively washed with glass distilled water, flooded with 0. 125% acidic Coomassie brilliant blue R-250 for 5 minutes, and then washed with water and coverslipped in 90% glycerol in PBS. Rhodamine-Phalloidin Staining of BPAE Cells For actin cytochemistry, cells were grown on 18 X 18 mm glass coverslips in MEM supplemented with 20% FCS. Treated and control monolayers were washed briefly with stabilization buffer (SB) (75 mM KCl; 3 mM MgSO4; 1 mM EGTA; 0.2 mM dithiothreitol; 10 mM imidazole; 10 jig/ml aprotinin; 1 X 10-4 M phenylmethylsulfonylfluoride, pH 7.2), at 37 C and permeabilized with 2 ml of 0.03% saponin (Sigma) in SB, pH 7.2, for 10 minutes at 20-22 C. Permeabilized monolayers were gently washed with SB and fixed in freshly prepared 3% formaldehyde in SB, pH 7.2, for 20 minutes at 20-22 C. Fixed monolayers were washed with SB and stained with rhodamine labeled phalloidin (Molecular Probes, Inc., Eugene, OR) for visualization of F-actin as outlined by Barak et al.'° Specimens were examined with a fluorescent microscope and photographed on Kodak Tri X film (ASA 400). Measurement of Actin Filament Content For determination of actin filament content of cells, we modified the procedure of Howard and Oresajo.'1""2 Cells were grown on 18 X 18 mm glass coverslips or in 35 X 10 mm Corning plastic culture dishes. Monolayers were washed with SB, permeabilized, and fixed as outlined above. Permeabilized monolayers were stained in the dark with 0.175 ,ug/ml rhodamine phalloidin for 30 minutes at 20-22 C. Monolayers were washed gently 3 times with SB and then flooded with 1 ml of ice cold methanol (HPLC grade). The monolayers were extracted with methanol for 30 minutes at -20 C and the endothelial cell cytoskeletons removed from the dish with a rubber policeman. Cul-

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WYSOLMERSKI AND LAGUNOFF

Figure 1-Untreated control monolayer of bovine pulmonary artery endothelial cells (BPAE) seven days postconfluent. Control monolayers were exposed to either PEG 400, DMSO/ethanol or TBS for 2 hours. Monolayers were stained with Eosin-Coomassie Blue as outlined in Materials and Methods. EC are tightly opposed to one another and the cell margins are prominently delineated by the Eosin Coomassie Blue staining. (X700)

ture dishes were washed with an additional 0.5 ml methanol, and the samples combined and stored at

-20 C for 12 hours for extraction ofrhodamine-phalloidin. Methanol extraction results in removal of 99% of the phalloidin bound to EC actin. The methanol suspension was centrifuged at 750g, the supernatant aspirated, and the precipitate allowed to air dry. The dry precipitate was dissolved in 6 N NaOH for protein determination. The rhodamine in the supernatants was determined using a fluorospectrophotometer (excitation 542 nm; emission 563 nm). The results are expressed as ng bound phalloidin/,ug protein. Actin filaments were removed from permeabilized EC by incubating monolayers in 0.3 M KI for 10 hours at 4 C. After KI incubation monolayers were washed with stabilization buffer, fixed, and stained as

AJP * July 1988

outlined above. KI extraction results in removal of 97% ofthe EC actin. Actin filament content was independently assessed by densitometry of SDS PAGE gels stained with Coomassie brilliant blue. Control and treated monolayers were prepared as outlined above. Dishes with attached EC cytoskeletons were flooded with 1 ml DPBS, pH 7.3, scraped with a rubber policeman, and the suspensions sonicated. Protein content of the suspensions was assayed. The cytoskeletal proteins were precipitated with 10 volumes ofice cold acetone at -20 C for 12 hours. The precipitates were air dried, resuspended in a small volume of SDS sample buffer (0.0625 M Tris HCl, pH 6.8; 10% glycerol; 2% SDS; 5% 2-mercaptoethanol and 0.01% bromphenol blue), and boiled for 3 minutes. SDS polyacrylamide electrophoresis was carried out in 5-15% vertical gradient mini slab gels using the buffer system of Laemmli.'3 Equal amounts of protein were applied to each well. The gels were stained with Coomassie brilliant blue R-250 and densitometry performed (LKB 2202 Ultroscan).

Special Reagents ECV and cyto B were prepared as described previously.6 A stock solution ofhistamine (disodium salt) was prepared at a concentration of 10 mM in a Trisma-buffered saline (TBS) (140 mM NaCl, 5.8 mM KCl, 3.0 mM CaCl2, 16.3 mM Trisma, pH 7.4). Histamine was prepared immediately before use at the desired working concentration in TBS containing 1% FCS or 1% BSA (Miles Path-o-cyte 4, final pH 7.4). Antimycin A (Sigma Chemical Co., St. Louis, MO) was prepared at a stock concentration of 10 mM in 100% ethanol and stored at -20 C until needed. Immediately before use the antimycin A was diluted in MEM without glucose and supplemented with 1% BSA and 2 mg/ml 2-deoxy-D-glucose (2 DG). Medium containing test reagents was warmed to 37 C and added to confluent monolayers for the desired time intervals. Control monolayers were incubated with media containing the appropriate diluent for equal times.

Results

Endothelial Cell Morphology Control monolayers formed a coherent sheet of Ir"L cells tightly apposed to one another with polygonal ~~~~~ cell margins identifiable with the Eosinprominent *k 'A'_ Coomassie blue (ECB) stain (Figure 1). Control BPAE cultures exposed to PEG 400, DMSO/ethanol or TBS Figure 2-BPAE monolayer exposed to 1 mg/ml ECV for 30 minutes. ECs have retracted from one another forming gaps between cells interrupting and stained with ECB showed no morphologic their previously linear prominent cell margins. Retracted endothelial cells remain attached to one another by slender filamentous processes. (x700) changes at any time interval studied. Monolayers ex-.r !

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ATP DEPLETION AND ENDOTHELIAL CELL RETRACTION

Figure 3-EC monolayer exposed to 500 AM histamine in TBS for 30 minutes. EC monolayers exposed to histamine exhibit widespread retraction from one another forming irregular gaps between adjacent cells. The prominent, continuous cell margins are lost; however, cells remain attached by filamentous processes. (x700)

Figure 4-Bovine pulmonary artery endothelial cells were exposed to 3 Ag/ ml cytochalasin B for 30 minutes. Severe widespread endothelial cell retraction occurs within 30 minutes. Wide gaps are present throughout the monolayer exposing large areas of the underlying culture dish. Cells remain attached to one another and their underlying matrix. (x700)

posed to 1 mg/ml ECV (Figure 2) for 30 minutes and then fixed and stained with ECB demonstrated moderate cell retraction with distinct gaps between adjacent endothelial cells. The gaps formed were bridged by numerous slender filamentous processes. With longer exposure to ECV, endothelial cell retraction increased as described previously.6 Cultures exposed to 500 ,uM histamine for 30 minutes (Figure 3) lost their prominent cell margins and exhibited mild retraction. Monolayers incubated in 3 ,ug/ml cyto B for 30 minutes exhibited extensive retraction ofthe cells with the formation of large gaps between adjacent cells (Figure 4). Cell-cell contact was largely lost. The cells remained adherent to one another through a small number of processes and to the underlying matrix.

with antimycin A or 2 DG alone, ATP levels dropped 14 and 21% respectively. When cells were exposed to antimycin A and 2 DG and the drugs removed, ATP levels recovered to 59% of control values after a 15 minute incubation in MEM + 10% FCS (Figure 7). There was little further recovery ofATP after an additional 60 minutes.

Cell ATP Content Cell ATP in control cultures was 44.7 ± 1.8 picomoles ATP/,ug protein. Continuous exposure of EC monolayers to 500 ,uM histamine, 1 mg/ml of ECV, or 3 ,tg/ml cyto B for time intervals ranging from 15 minutes to 1.5 hours, resulted in no significant reduction of ATP content (Table 1). In preliminary experiments, dinitrophenol, sodium azide, antimycin A, and 2 DG were tested for their effects on cell ATP. The combination of antimycin A and 2 DG was found to be particularly effective in reducing ATP content. When monolayers were incubated with glucose-free MEM containing 1% BSA, 25 nM antimycin A, and 2 mg/ml 2 DG, ATP levels rapidly declined over a period of 60 minutes to 5% of the original levels (Figure 7). When glucose was included in the medium, ATP levels were reduced 35% in the same period. In monolayers incubated for 60 minutes

Effects of ATP Depletion EC cultures depleted of greater than 90% of their ATP content when fixed and stained with ECB exhibited only minor morphologic alterations (Figure 5). There was diminution in the prominence of the ECB staining of cell margins, but only rare gaps were detected between adjacent cells. Neither cell rounding nor detachment from the culture dishes was observed. The normal prominence of the cell margins demonstrable with ECB staining was not re-established for several hours after the removal of the inhibitors. When ATP-depleted cultures were exposed to either 1 mg/ml ECV, 500 ,uM histamine, or 3 ,g/ml cyto B for as long as 60 minutes, no retraction occurred (FigTable 1 -Effect of Histamine, ECV and Cyto B on Cellular ATP Levels

Time (minutes)

ATP content*

0

15

30

60

90

Histamine 44.7 t 1.8 43.2 ± 0.9 45.4 ± 1.9 42.5 ± 1.9 ND ECV 44.7 ± 1.8 ND 44.7 ± 2.6 44.3 ± 3.9 44.9 ± 2.2 44.7±1.8 44.7±1.8 43.8±1.7 44.1 ± 1.8 CytoB ND ^ ATP values expressed as picomoles ATP//lg protein. Determination of cellular ATP content was performed as outlined in Materials and Methods. Protein per cell was not affected by any of the agonists. ND, not determined.

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WYSOLMERSKI AND LAGUNOFF

AJP . July 1988 50

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Figure 5-EC monolayer exposed to 25 nM antimycin A + 2 mg/ml 2-deoxyglucose in the absence of glucose for 60 minutes. Decreased prominence of EC margins is the major morphologic manifestation of reduced ATP levels. Occasional focal gaps between adjacent cells can be encountered. (X700)

ure 6). Only an occasional gap was encountered between cells as a result of ATP depletion itself. When ATP deficient monolayers were allowed to partially recover their ATP content, the ability of endothelial cells to retract was restored.

Distribution of F-actin The distribution of actin filaments identified by rhodamine-phalloidin staining in untreated BPAEC was rather complex as described previously.6 There was a prominent dense peripheral band of actin, a limited array of filament bundles oriented parallel to one another and to the long axis of the cell, and a complex arrangement ofparanuclear filamentous actin (Figure 8). The peripheral band of actin was highly consistent

Figure 6-ATP depleted monolayer exposed to 1 mg/ml ECV for 15 minutes in the continued presence of inhibitors of ATP production. Reduction of EC ATP levels inhibits ECV, cyto B and histamine induced endothelial cell retraction. No significant differences in ATP-depleted drug treated (Figure 6) monolayer can be detected from ATP-deplete monolayer treated with drug carrier (Figure 5). (x700)

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MINUTES Figure 7-Time course of ATP depletion and recovery (*) in bovine pulmonary artery endothelial cells. EC were incubated in glucose-free media containing either antimycin A + 2-deoxyglucose (A --A), antimycin A alone (0- 0), or 2-deoxyglucose (A - A), for the desired intervals and analysis of cellular ATP content performed as outlined in Materials and Methods. EC incubated with MEM containing glucose and 10% FCS resulted in recovery of cellular ATP content (*). Each point is mean ± SD of 5 separate experiments.

from cell to cell whereas the other distributions of actin filaments were variable from cell to cell. The effects of ECV on F-actin distribution have been previously described.6 Briefly, ECV causes the loss of F-actin from the dense peripheral band and a reorganization of paranuclear filaments into a prominent series of filaments aligned parallel to each other and to the long axis of the cell. In BPAE monolayers exposed to 500 ,uM histamine for 5 minutes (Figure 9A) there was focal loss of F-actin staining from the dense peripheral band, but the central microfilament

Figure 8-Fluorescent F-actin distribution in control BPAE cell monolayers 7 days postconfluent. EC margins are delineated by the prominent dense peripheral band (DPB) of F-actin filaments. Central microfilament bundles traverse the long axis of the cell interconnecting with the paranuclear array of actin filaments. (x500)

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ATP DEPLETION AND ENDOTHELIAL CELL RETRAMTON

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Figure 9A-EC monolayer exposed to 500 AM histamine for 5 minutes. Selected areas of endothelial cell retraction are evident with loss of the peB-Widespread rearrangement of the ripheral band of actin. (x500) dense peripheral band has occurred after 20 minutes exposure to 500 AM histamine. The dense peripheral band of actin filaments retracts assuming a prominent subcortical distribution. Central microfilament bundles remain intact. (x500)

Figure 10-Loss of the dense peripheral band of actin filaments results from exposure of monolayers to 25 nM antimycin A + 2 mg/ml 2-deoxyglucose for 60 minutes in glucose-free media. The central microfilament bundles have lost their regular organized distribution. Only remnants of the paranuclear array of actin filaments remain intact. The central microfilament bundles are in disarray and puddles of fluorescent staining material can be found scattered within the cytoplasm of some cells. (x500)

bundles were unchanged. At 20 minutes (Figure 9B), the dense peripheral band of actin filaments had retracted to a subcortical position; the central microfilament bundles and paranuclear actin array remained intact. Monolayers exposed to 3 ,g/ml cyto B for 30 minutes (Figure 15) lost the dense peripheral band and much of the central microfilament array. Only scattered microfilament bundles were evident in a few cells. Droplets of F-actin were regularly present along the cell margins, particularly at sites of persisting cellcell attachment and occasionally elsewhere within the cytoplasm (Figure 15). On reduction of ATP, the F-actin network underwent time-dependent rearrangements. Monolayers with ATP reduced to 10% or less of control levels exhibited extensive loss of the dense peripheral band with some loss of the paranuclear array. In many cells, droplets of F-actin appeared throughout the cytoplasm. The central microfilaments were in disarray and frequently appeared wavy. By 60 minutes, the dense peripheral band of actin was lost entirely and only remnants of the paranuclear array remained (Figures 10, 1 1), replaced by a disorganized, tangled central web of filaments and scattered droplets of Factin. On removal of the antimycin A and 2 DG, the reestablishment of the original actin distribution paralleled the recovery of cell ATP content. By 15 minutes (Figure 12) actin cables appeared at the periphery of the cells and the paranuclear array of actin began to reform. By 60 minutes, the dense peripheral band began to reform, and the paranuclear array of actin fil-

aments and the longitudinal actin bundles were largely restored. Four hours after initiation of reversal of ATP depletion, re-establishment of the original actin cytoskeleton was virtually complete (Figure 13). Neither ECV nor histamine had any apparent effect on the already perturbed actin distribution in ATPdepleted endothelial cells (Figure 14A, 14B). Although ATP-depleted monolayers exposed to cyto B underwent only slight morphologic changes, F-actin distribution was further modified with the loss of central microfilament bundles and the appearance of

Figure 11-Enlarged view of ATP depleted BPAE cell. The dense peripheral band of actin is lost. Central microfilaments are disorganized and droplets of F-actin are present within the cytoplasm. The central bundles of filaments appear wavy with occasional curled ends. (x1200)

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AJP . July 1988

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Figure 12-F-actin distribution in ATP depleted monolayers allowed to recover for 15 minutes in complete growth media. Recovery of cellular ATP results in the reestablishment of the DPB as well as reorganization of the central microfilament bundles. After 15 minutes recovery the majority of central actin bundles are again traversing the long axis of the cell interconnecting with the reformed paranuclear array of actin filaments. (x500)

Figure 14A-Fluorescent actin distnibution in monolayers depleted of ATP and then exposed to 1 mg/ml ECV for 30 minutes in the presence of inhibitors of energy metabolism. No change in F-actin can be detected from those changes that result from ATP depletion alone. (x500) B-ATP depleted monolayers incubated with 500M,M histamine in the presence of 25 nM Antimycin + 2 mg/mI 2DG. No additional redistribution of F-actin cytostructure from that observed from ATP depletion could be detected. (x500)

prominent irregular accumulations of fluorescent staining beneath the plasma membrane (Figure 16).

tion of cytoskeletal actin. Histamine or ECV exposure resulted in only a small reduction in F-actin content (6 and 8% respectively), whereas in response to cyto B there was a 50% decline in filamentous actin within 15 minutes (Figures 17, 18, and 19). A 19% decline in endothelial cell F-actin occurred in response to ATP

F-actin Quantitation In view of the changes in actin distribution caused by ECV, histamine, and cyto B and ATP depletion it was of interest to measure the F-actin in each instance. Filamentous actin content was assessed both by methanol extraction of rhodamine-phalloidin bound to the cytoskeleton and by gel electrophoretic quantita-

Figure 13-Four hours after washout of antimycin A + 2-deoxyglucose the EC F-actin distribution appears similar to that of control. The DPB has reformed and the central microfilaments traverse the long axis of the cell interconnecting with the paranuclear array of actin filaments. (x500)

depletion (Figure 17). Treatment of monolayers depleted of ATP with either histamine or ECV resulted in no further reduction of F-actin content (Table 2). Cyto B treatment of

ATP-depleted cultures resulted in an additional re-

Figure 15-Actin filament distribution in BPAE monolayers 30 minutes after exposure to 3 ug/ml cyto B. Severe endothelial retraction results within 30 minutes, with the loss of the dense peripheral band of actin and most of the central microfilament bundles. Aggregates of fluorescent material are present at sites of cell attachment and along the cell margins. (x500)

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ATP DEPLETION AND ENDOTHELIAL CELL RETRACTION

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Figure 16-Fluorescent micrograph of ATP depleted monolayer exposed to 3 Mg/ml cytochalasin B for 15 minutes. A marked loss of central microfilament bundles resulted from cyto B exposure in comparison to the effect of ATP depletion alone (cf. Figures 10, 11). A prominent peripheral staining results delineating the cell margins. (X500)

duction in filamentous actin (Table 2) to 38% of the control level in 30 minutes, consistent with a nearly additive effect of ATP and cyto B.

Discussion A critical function of the endothelium is to serve as a permeability barrier separating intravascular contents from the extravascular compartment. A break in this barrier results in edema. Although numerous

studies using well-characterized inflammatory mediators in vivo1"2"14'20 and in vitro'5-7 have been performed, the mechanism responsible for increased permeability remains unidentified. Majno and Palade'4 described the opening of interendothelial junctions in the in vivo response to histamine, and Majno et al'8 proposed subsequently that active contraction of endothelium affected through cytoskeletal elements was responsible for the formation of gaps between cells. While this proposal has had considerable appeal, direct evidence for the contractile basis of gap formation has been limited. We6 and others' 57"9'28 have implicated changes in the cytoskeleton in endothelial cell retraction in vitro induced by agents capable of causing edema. The most obvious model for endothelial cell retraction involves a contractile event in which a network of actin filaments is modified by the action of myosin.2022 Depletion of ATP could prevent retraction according to this model through its role in either stabilizing F-actin,23'24 serving as a substrate for myosin ATPase,25,26

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MINUTES Figure 17-Time course of changes in EC F-actin content after exposure to ECV (O-0), histamine (A - A), cyto B (A --*), and antimycin A + 2DG (O- l). Assessment of EC F-actin content was performed as outlined in Materials and Methods. Each point is mean ± SD of 6 separate experiments.

or phosphorylating myosin light chains.27 In the present study we performed a series of experiments to determine if ATP is essential for cell retraction and to examine the effect of the retraction-inducing agents as well as the reduction of cell ATP levels on F-actin content and distribution. Reduction of ATP prevented retraction by all 3 agents. Our previous experiments indicated that retraction is consistently accompanied by a rearrange400 r

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MINUTES Figure 18-Time course of changes in F-actin in EC cytoskeletons. EC were exposed to ECV (O-0), histamine (A - A) and cyto B (CB, A - A) for the times indicated, permeabilized with 0.03% saponin and processes as outlined in Materials and Methods. Saponin insoluble cytoskeletons were run on 5-15% SDS polyacrylamide gels and changes in actin content assessed by densitometry.

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WYSOLMERSKI AND LAGUNOFF

AJP * July 1988

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