Histamine, actin-gelsolin binding, and polyphosphoinositides in human umbilical vein endothelial cells MARK R. CARSON, SANDRA S. SHASBY, STUART E. LIND, AND D. MICHAEL SHASBY Department of Medicine, University of Iowa College of Medicine, and the Veterans Affairs Hospital, Iowa City, Iowa 52242; and Divisions of Hematology-Oncology and Experimental Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02215 Carson, Mark R., Sandra S. Shasby, Stuart E. Lind, and D. Michael Shasby. Histamine, actin-gelosin binding, and polyphosphoinositides in human umbilical vein endothelial cells. Am. J. Physiol. 263 (Lung Cell. Mol. Physiol. 7): L664L669, 1992.-Histamine activates inositol phospholipid metabolism, increases calcium, and causes a change in shape of human umbilical vein endothelial (HUVE) cells. Changes in endothelial cell shape are determined, in part, by changes in the actin cytoskeleton. Gelsolin is an actin-binding protein with the potential to alter the actin cytoskeleton in response to changes in cell calcium and/or changes in polyphosphoinositides. Therefore, we examined the interactions of actin and gelsolin in HUVE cells in which inositol phospholipid metabolism was activated with histamine. In HUVE cells exposed to histamine we estimated actin-gelsolin binding by quantitating actin and gelsolin, immunoprecipitated with anti-gelsolin Sepharose. We estimated the relative amount of filamentous actin in the histamine-exposed HUVE cells by quantitating the amount of actin that was Triton soluble. We also measured the amount of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5bisphosphate (PIPB) in the HUVE cells before and after exposure to histamine. We found that histamine decreased the amount of actin that was immunoprecipitated with gelsolin, decreased the fraction of cell actin that was Triton soluble, and increased PIP and PIP,. These results demonstrate that histamine promotes actin filament formation in HUVE cells and that histamine-mediated changes in actin-gelsolin binding in these cells are better predicted by changes in polyphosphoinositides than by increases in cell calcium. histamine; endothelium
MAJNO AND PALADE (20) first reported that adjacent endothelial cells separated from one another when the endothelium was exposed to the edemagenic molecules histamine or serotonin. On the basis of their electron microscopic observations, they suggested that the cells became disconnected along their intercellular junctions. Since then several authors (I, 14, 17, 25-27, 32) have described the development of gaps between adjacent endothelial cells when the endothelium has been exposed to agents that cause edema. Although there are multiple reports of gaps developing between endothelial cells in the setting of edema, the mechanism of the change in endothelial cell shape that causesthe gaps remains poorly understood. Agents that break down actin filaments or that disrupt cell-cell or cell-substrate attachments cause edema and gap formation (21, 26, 27). This suggests that the gaps may result from loss of tethering of cells to each other or to substrate. However, the change in endothelial cell shape caused by histamine is dependent on cell ATP (30). This suggeststhat gap formation in response to histamine is, at least in part, an energy-dependent process. Consistent with this, two groups of investigators found that retraction of the actin cytoskeleton of permeabilized endotheL664
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lial ceils was dependent on activation of myosin lightchain kinase (24, 31). It is conceivable that all of these processes, i.e., release of tethering, changes in actin filaments, and active contraction, contribute to the development of gaps between adjacent endothelial cells exposed to edemagenic molecules. Gelsolin is an actin-binding protein that contributes to changes in cell shape by altering the actin cytoskeleton (6). In the presence of micromolar calcium, gelsolin severs actin filaments and binds to the barbed end of the actin filament, preventing filament elongation. However, gelsolin also promotes actin filament assembly by providing nuclei of filamentous actin (F-actin), which can rapidly assemble into filaments when gelsolin is displaced from actin by the polyphosphoinositides, phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) (6). One of the immediate effects of histamine binding to an H1 receptor on endothelial cells is initiation of inositol phospholipid metabolism and a consequent increase in cell calcium (3, 23). Hence, binding of histamine to an Hi receptor on endothelial cells would likely result in changes in actin-gelsolin interactions, either because of changes in cell calcium or because of changes in polyphosphoinositides, or because of changes in both. In these studies we examined whether histamine activation of signal transduction affected actin-gelsolin interactions in human umbilical vein endothelial (HUVE) cells. METHODS
Materials. Histamine, piperazine-N-N’-bis(2-ethanesulfonic acid) (PIPES), N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES), EGTA, leupeptin, benzamide, aprotinin, phenylmethylsulfonyl fluoride (PMSF), Triton X-100, Na ATP, glucose, and electrolytes were from Sigma Chemical, St. Louis, MO. Monocolonal anti-gelsolin antibodies were conjugated to Sepharose beads as described (18). The specificity of these antibodies has been detailed (4). Tissue culture media and supplies were from GIBCO via the Cancer Center, University of Iowa. Selectamine was from GIBCO. Collagenase was from Worthington Biochemical, Freehold, NJ. Fetal bovine serum (FBS) was from Hyclone, Logan, UT. [““S]methionine(70%)-cysteine(30%) was from ICN, Irvine, CA. [3H]inositol was from New England Nuclear (Boston, MA). Cell culture. HUVE were prepared by collagenase treatment of freshly obtained umbilical veins (3). Harvested cells were plated on 60-mm-diam tissue culture plates (Costar). Cells were cultured in medium 199 (M-199) with 20% FBS, basal medium Eagle vitamins and amino acids, glucose (5 mM), glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 pg/ml). All studies were conducted on primary cu?ures. Such cultures were identified as endothelial by characteristic uniform morphology, uptake of fluorescently labeled acetylated low-density lipo-
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protein, and indirect immunofluorescent staining for factor VIII (3). Cell proteins were labeled with [“5S]methionine by changing the medium 1 day prior to confluence to Selectamine (methionine free) supplemented with 10% FBS and 180 &i/ml [35S]methionine and incubating for 48 h. Labeled cells were then exposed to histamine or vehicle [Hanks’ balanced salt solution (HBSS)] for the indicated times, the labeling media were removed, and the cells were immediately snap frozen on dry icemethanol and then thawed in the lysing buffer as described in Determination of actin-gekolin ratios. A histamine dose of 1 X 10e5 M was used based on our earlier report (3). Cell inositol phospholipids were labeled by adding 4 PCi [3H]inositol to the M-199-20% FBS medium and incubating them for 72 h. After the 72 h, the plates were rinsed with HBSS and then exposed to histamine or vehicle (HBSS) (3). Determination of actin-gelsolin ratios. Cells labeled with [35S]methionine were exposed to histamine or vehicle for the indicated times, the labeling media were removed, and the cells were snap frozen on dry ice-methanol. One milliliter of lysing buffer (120 mM PIPES, 50 mM HEPES, 20 mM EGTA, 4 mM MgClz, 10 mM glucose, 20 kg/ml leupeptin, 156 pg/ml benzamide, 80 pg/ml aprotinin, 1 mM PMSF, and 1.5% Triton X-100, pH 7.2) was added to the plate, and the cells were thawed in the lysing buffer (18). The plates were scraped, and the lysates were centrifuged at 12,000 g for 10 min. Eight hundred microliters of the supernatant solution were added to 30 ~1 of a 50% suspension of anti-gelsolin-Sepharose beads and rotated at 4°C for 2 h, and then the beads were pelleted by centrifugation at 12,000 g for 2 min. The pelleted beads were then washed with 1 ml lysing buffer with 5 mM NaATP, repelleted, and then incubated for 15 min at room temperature in 0.3 M MgC12, 1 mM EGTA, 50 mM tris(hydroxymethyl)aminomethane (Tris) and 100 mM NaCl, pH 7.4, to depolymerize actin filaments associated with the beads (18). After repelleting, the beads were washed with the same solution without the MgClz. Washed beads were boiled in gel sample buffer and loaded onto 5-15% polyacrylamide gels (15). The gels were electrophoresed at 90 V for 15 h, dried, and incubated with X-ray film at -70°C for 60 h. The autoradiograms were developed, and the relative densities of actin and gelsolin were determined by laser densitometry (Molecular Dynamics) . Phospholpid analyses. [3H]inositol (4 mC) in M-199 with 20% FBS was added to each tissue culture plate for 72 h. After the 72 h the medium was removed, and the plates were washed three times with HBSS. HBSS (2 ml) was then added to each plate, and each plate was then exposed to HBSS or histamine (1 x 10m5M). After the indicated time, the medium was removed, the plates were snap frozen on dry ice-methanol, and the cell lipids were extracted according to Bligh and Dyer as previously described (2,3). The chloroform layer was dried under nitrogen, dissolved in chloroform-methanol (2:1), and spotted on preactivated Silica gel 60 plates. The plates were developed in chloroform-methanol-methyl amine-2.4 N HCl (60:35:5:5) (35). Radioactivity in individual lipid fractions was quantitated on a Radiomatics Instruments thin-layer gel scanner (model RS). Standards for phosphatidylinositol, PIP, PIP,, and lysophosphatidylinositol were identified with iodine vapor. In two experiments, cell phospholipids from the chloroform phase of the extraction were spotted onto the same Silica gel plates and separated as above. The inositol phospholipids were identified with standards and scraped into 1.8 ml acid saline for determination of inositol lipid phosphate as described (28). An aliquot of the chloroform phase from the initial Bligh-Dyer extraction was used to determine total cell lipid phosphate. Analysis of Triton-soluble actin. To measure Triton-soluble actin, cytoskeletal proteins were extracted as described by Isaacs and Fulton (10). Briefly, HUVE cells in 60-mm-diam
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tissue culture plates were rinsed free of serum-containing medium and then exposed to HBSS with or without 1 x 10e5 M histamine for the indicated times. Cells were then extracted with 4°C skeleton buffer ( 100 mM KCl, 10 mM PIPES, pH 6.8, 300 mM sucrose, 2 mM MgClz, 1 mM EGTA) with 0.5% Triton X-100, 1 mM PMSF, 100 FM leupeptin, 5 mM benzamide, 5 mM c-aminocaproic acid, and 2 mM iodoacetamide for 10 min. A second extraction with same buffer and protease inhibitors was done at room temperature. These extractions were combined and centrifuged at 100,000 g for 15 min at 4°C. The supernatant was removed and defined as the Triton-soluble skeleton. The cytoskeleton remaining on the tissue culture plate was then extracted with skeleton buffer containing 2% sodium dodecyl sulfate, 0.5% 2-mercaptoethanol, and 100 mM NaCl instead of KCl, sheared through a 26-gauge needle, and the nucleic acids were precipitated by centrifugation at 100,000 g for 15 min. The supernatant from this centrifugation was combined with the precipitate from the Triton extractions and defined as the Triton-insoluble fraction. Aliquots from the Triton-soluble and -insoluble fractions were dissolved in gel sample buffer and chromatographed on 8-15% polyacrylamide gradient gels (15). The gels were stained with Coomassie Blue and dried, and actin content was quantitated by laser densitometry (Molecular Dynamics). Statistical analyses. All data are presented as means f SE. An individual number (n) represents a tissue culture plate. Differences among groups were assessed by analysis of variance with differences between individual groups determined by Tukey highly significant difference test for post hoc comparisons of means. Groups that are referred to as different (greater than or less than) were statistically different at P 5 0.05. RESULTS
Actin-to-gelsolin ratios. After HUVE cells were exposed to histamine, the amount of actin that was immunoprecipitated from the cells with anti-gelsolin antibody decreased relative to the amount of gelsolin that was similarly immunoprecipitated. Figure 1 is an autoradiogram of [35S]methionine-labeled HUVE cell proteins immunoprecipitated with anti-gelsolin antibody coupled to Sepharose beads. Lane 1 is from cells not exposed to
116,250 97,400 66,200
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31,000 Fig. 1. Autoradiogram of [3”S]methionine-labeled proteins immunoprecipitated from human umbilical vein endothelial (HUVE) cells with anti-gelsolin Sepharose. Lane 1, control cells; lane 2, cells exposed to histamine (1 X 10m5M) for 45 s; lane 3, cells exposed to histamine for 90 s.
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histamine, lane 2 is from cells exposed to histamine for 45 s, and lane 3 is from cells exposed to histamine for 90 s. The band at 91 kDa is gelsolin, and the band at 42 kDa is actin. The large band at 56 kDa is believed to be vimentin, based on molecular weight and its abundance in endothelium. After HUVE cells were exposed to histamine, the density of the actin band relative to the gelsolin band rapidly decreased (0.43 t 0.04 at 45 s vs. 0.60 t 0.04 in control). Figure 2 presents a summary of the relative densities of the actin and gelsolin bands immunoprecipitated from [ 35S] methionine-labeled HUVE cells before (Con) and after exposure to histamine for 90 s and 5 min. Triton-soluble actin. The decrease in actin-gelsolin binding observed after exposing HUVE cells to histamine might be expected to enhance the incorporation of actin into the filamentous cytoskeleton (13). To assess this we measured the fraction of actin that was Triton soluble, before and after exposing HUVE cells to histamine. Figure 3 demonstrates that, in HUVE cells exposed to histamine, the fraction of actin that was Triton soluble decreased by 90 s and remained decreased at 5 min. Inositolphospholipids. PIP and PIP, dissociate gelsolin from the barbed end of the actin filament. Because histamine decreased actin-gelsolin binding, it was of interest to determine if histamine increased PIP and PIP, levels in HUVE cells. The mass of total inositol lipid phosphate did not change after HUVE cells were exposed to histamine (Fig. 4). However, the fraction of inositol phospholipid that was present as PIP or PIP2 did increase after HUVE cells were exposed to histamine. Figure 5, A and B, are tracings from the thin-layer gel scanner, demonstrating relative peak areas for PIP2 (peak 1), PIP (peak 2) and PI (peak 3) from [3H]inositol-labeled HUVE cells before (A) and after (B) exposure to histamine. Ninety seconds after HUVE cells were exposed to histamine, PIP had increased as a percent of inositol phospholipid, and by 5 min both PIP and PIP2 had increased as a percent of inositol phospholipid (Fig. 6). Because total inositol phospholipid mass was constant after exposure to histamine, the mass of PIP and PIP2 increased in HUVE cells exposed to histamine. DISCUSSION
Actin is an important constituent of the cortical cytoskeleton of epithelioid cells, where it is linked to spectrin and myosin and to the receptors for cell-cell contact 0.8 .I 5
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.Is 5 v) 0.4 Q) s? s 'g 0.2 a
90 Set 5 Min Fig. 2. Ratios of densities of bands of actin to bands of gelsolin immunoprecipitated from HUVE cells before (Con) and after exposure to histamine for 90 s and 5 min. n = 8 for each time. P 5 0.05 for 90 s and 5 min compared with control.
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Control ’ 90 Set ’ 5 Min ’ Fig. 3. Fraction of actin that was Triton soluble in HUVE cells before and after exposure to histamine for 90 s and 5 min. n = 4 for each time. P 5 0.05 for 90 s and 5 min compared with control.
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Fig. 4. Inositol lipid phosphate in HUVE cells before and after exposure to histamine for 90 s and 5 min. n = 6 for each time. There were no differences in inositol lipid phosphate at different times.
and cell-substrate contact (18, 21). Molecules such as the cytochalasins, which alter the physiochemical state of actin, alter epitheliod cell shape and the integrity of epithelial and endothelial barriers (19, 27). Edemagenic molecules such as histamine alter endothelial cell shape, and it is likely that histamine-mediated changes in endothelial cell shape involve changes in actin in endothelial cells. Gelsolin is an actin-binding protein that can shorten actin filaments and block filament assembly when it binds to the barbed end of actin filaments. Dissociation of gelsolin from the barbed end of actin filaments can also promote rapid filament assembly by providing a large number of oligomers with a free barbed end (6, 29). The binding of gelsolin to actin is stimulated by micromolar calcium and inhibited by PIP and PIP2 (33, 34). When macrophages and platelets are activated, gelsolin approaches the plasma membrane, where it could be exposed to high local concentrations of these polyphosphoinositides. Because binding of histamine to the H1 receptor on endothelial cells activates inositol phospholipid metabolism and increases calcium, it was likely that histamine would alter actin-gelsolin interactions in endothelial cells (3). We found that within 45 s of adding histamine to [35S]methionine-labeled HUVE cells, the relative densities of the actin/gelsolin bands decreased from control levels of binding is 0.6 to -0.4. This decrease in actin-gelsolin similar to the decrease in actin-gelsolin binding found by Dadabay et al. (7) in A 431 cells exposed to epithelial growth factor (EGF). Dissociation of actin from gelsolin would be expected to
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10 8 6 4 2 0 Con Fig. 6. Distribution labeled HUVE cells mine (1 X low5 M); min compared with control.
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Fig. 5. A: tracing of thin-layer chromatogram from [3H]inositol-labeled HUVE cells before exposure to histamine. Peak 1 represents phosphatidylinositol 4,5bisphosphate (PIP2), peak 2 represents phosphatidylinositol 4-phosphate (PIP), and peak 3 represents phosphatidylinositol. B: tracing of similar thin-layer chromatogram from HUVE cells 5 min after exposure to histamine (1 X 10e5 M).
increase incorporation of actin into microfilaments. Cell actin is frequently functionally divided into the Tritonsoluble (nonfilamentous) and -insoluble (filamentous) fractions for purposes of estimating how much of the actin is incorporated into microfilaments (10). We found that the fraction of actin that was Triton insoluble increased after histamine exposure, consistent with the decrease in actin-gelsolin binding. This increase in F-actin is consistent with the findings of Dadabay et al. (7), who found that EGF increased F-actin in A 431 cells, and with those of Eberle et al. (8), who found that N-formyl-MetLeu-Phe increased actin polymerization in neutrophils. Because histamine rapidly increases endothelial cell calcium, it was surprising that the initial response was not an increase in actin-gelsolin association. Histamine increases endothelial cell calcium to -500 nM, whereas the affinity constant of gelsolin for calcium is 920 nM (6, 33, 34). Hence, histamine may not increase endothelial cell calcium enough to increase actin-gelsolin binding. However, since our measurements represent the net average actin-gelsolin binding in the whole cell, our data do
90 Set
of counts in inositol before and 90 s and n = 9 for each time. control. P 5 0.05 for
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phospholipids in [3H]inositol5 min after exposure to histaP 5 0.05 for PIP at 90 s and 5 PIP2 at 5 min compared with
not exclude the possibility that actin-gelsolin binding increases within specific cellular regions. There have been two independent reports that calciummediated changes in the shape of the actin cytoskeleton of detergent-permeabilized endothelial cells is dependent on activation of phosphorylation of the light chain of myosin (24, 31). Phosphorylation of the light chain of myosin is important for the maintenance and assembly of actin microfilaments in nonmuscle cells (16). Both phosphorylation of the myosin light chain and dissociation of actin from gelsolin would enhance microfilament assembly. Hence, the effects of histamine on actin-gelsolin would complement effects of histamine on calcium-initiated myosin light chain phophorylation in endothelium and promote the observed increase in F-actin. In addition to increasing endothelial cell calcium, we found that histamine increased endothelial cell PIP and PIP2. In vitro, increased concentrations of PIP or PIP2 decrease actin-gelsolin binding and promote actin filament assembly (1 l- 13). In activated macropha .ges and platelets, actin- #gelsolin complexes associate with the cell membrane where they would be exposed to high local concentrations of PIP and PIP2 (9). Based on the these observations, the observed histamine-initiated increases in PIP and PIP2 may be causally related to the detected decreases in actin-gelsolin binding. Whereas Dadabay et al. (7) found that EGF decreased actin-gelsolin binding in A 431 cells, their data did not support the hypothesis that changes in PIP and PIP,, might cause the decreased binding. However, there are some important differences between A 431 and HUVE cells and their responses to the respective agonists. EGF increased PIP in A 431 cells but, unlike HUVE cells stimulated with histamine, EGF did not increase, but actually decreased PIP2 in A 431 cells. Hence, there is a difference in inositol metabolism between the cells after
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stimulation with the respective agonists. Additionally, cholera toxin pretreatment prevented the EGF-stimulated increase in PIP but did not prevent membrane ruffling or the decrease in actin-gelsolin binding in A 431 cells (7). In contrast, pretreatment of HUVE cells with agents which increase CAMP, prevents cell retraction and myosin light chain phosphorylation, both of which imply major effects of CAMP on F-actin (Refs. 3 and 15 and A. Moy and D. M. Shasby, unpublished observations). Hence, in HUVE cells it would not be possible to independently interpret effects of CAMP on the actin cytoskeleton and on inositol phospholipid metabolism. Eberle et al. (8) observed that increased F-actin in N-formyl-Met-Leu-Phe-activated neutrophils was associated with an increase in phosphatidylinositol trisphosphate (PIPS) in the neutrophils. We did not measure PIP3 in HUVE cells exposed to histamine and therefore cannot comment on the relevance of Eberle’s observations to ours. However, in the neutrophils, PIP2 decreased after activation, in contrast to the increase in PIP2 in HUVE cells exposed to histamine. Whether some of the increase in PIP2 measured in HUVE cells exposed to histamine represents comigration of PIP2 and PIP3 is not possible for us to determine at this time. In summary, histamine changes endothelial cell shape, and the change in cell shape likely involves changes in the actin cytoskeleton. Gelsolin is an actin-binding protein with the capacity to profoundly alter the actin cytoskeleton in response to calcium or in response to polyphosphoinositides. In HUVE cells activated with histamine, gelsolin interactions with actin are better predicted by changes in HUVE cell polyphosphoinositides than by the increases in cell calcium. This research was completed during D. M. Shasby’s tenure as a Clinical Investigator of the Veteran’s Administration. The work was also supported by National Heart, Lung, and Blood Institute Grant HL-33540. Address for reprint requests: D. M. Shasby, Dept. of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA, 52242. Received
7 May
1991; accepted
in final
form
10 August
1992.
REFERENCES 1. Albertine, K. H., J. P. Weiner-Kronish, K. Koike, and N. C. Staub. Quantification of damage to lung microvessels in anesthetized sheep. J. Appl. Physiol. 57: 1360-1368, 1984. 2. Bligh, E. G., and W. J. Dyer. A rapid method of total lpid extraction and purification. Can. J. Biochem. 37: 911-917, 1959. 3. Carson, M. R., S. S. Shasby, and D. M. Shasby. Histamine and inositol phosphate accumulation in endothelium: CAMP and a G protein. Am. J. Physiol. 257 (Lung Cell. Mol. Physiol. 1): L259L264, 1989. 4. Chaponnier, C., P. A. Janmey, and H. L. Yin. The actin filament-severing domain of plasma gelsolin. J. CeZZ Biol. 103: 1473-1481, 1986. 5. Chaponnier, C., H. L. Yin, and T. P. Stossel. Reversibility of gelsolin. Actin interaction in macrophages: evidence for Ca2+ dependent and Ca2+ independent pathways. J. Exp. Med. 165: 97106, 1987. 6. Cunningham, C. C., T. P. Stossel, and D. J. Kwiatowski. Enhanced motility in NIH 3T3 fibroblasts that overexpress gelsolin. Science Wash. DC 251: 1233-1236, 1991. 7. Dadaby C. Y., E. Patton, J. A. Cooper, and L. J. Pike. Lack of correlation bewteen changes in polyphosinositide levels and actin/gelsolin complexes in A 431 cells treated with epidermal prowth factor. J. CeZZ BioZ. 112: 1151-1156. 1991.
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8. Eberle, M., A. E. Traynor-Kaplan, L. A. Sklar, and J. Norgauer. Is there a relationship between phosphatidykinositol trisphosphate and F-actin polymerization in human neutrophils? J. BioZ. Chem. 265: 16725-16728, 1990. 9. Hartwig, J. H., K. A. Chambers, and T. P. Stossel. Association of gelsolin with actin filaments and cell membranes of macrophages and platelets. J. CeZZ BioZ. 108: 467-479, 1989. 10. Isaacs, W. B., and A. B. Fulton. Cotranslational assembly of myosin heavy chain in developing cultured skeletal muscle. Proc. Natl. Acad. Sci. USA 84: 6174-6178, 1987. 11. Janmey, P. A., K. Iida, H. L. Yin, and T. P. Stossel. Polyphosphoinositide micelles and polyphosphoinositide-containing vesicles dissociate endogenous gelsolin-actin complexes and promote actin assembly from the fast-growing end of actin filaments blocked by gelsolin. J. BioZ. Chem. 262: 12228-12236, 1987. 12. Janmey, P. A., and T. P. Stossel. Modulation of gelsolin function by phosphatidylinositol 4,5-bisphosphate. Nature Lond. 325: 362-364, 1987. 13. Janmey, P. A., and T. P. Stossel. Gelsolin-polyphosphoinositide interaction. J. Biol. Chem. 264: 4825-4831, 1989. 14. Kadish, J. L., C. E. Butterfield, and J. Folkman. The effect of fibrin on cultured vascualr endothelial cells. Tissue CeZZ 11: 99-108, 1979. 15. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature Lond. 227: 680685, 1970. 16. Lamb, N. J. C., A. Fernandez, M. A. Conti, R. Adelstein, D. B. Glass, W. J. Welch, and J. R. Feramisco. Regulation of actin microfilament integrity in living nonmuscle cells by the CAMP-dependent protein kinase and the myosin light chain kinase. J. CeZZ BioZ. 106: 1955-1971, 1988. 17. Laposata M., D. K. Dovnarsky, and H. S. Shin. Thrombininduced gap formation in confluent endothelial cell monolayers in vitro. BZood 62: 549-556, 1983. 18. Lind, S. E., P. A. Janmey, C. Chaponnier, T.-J. Herbert and T. P. Stossel. Reversible binding of actin to gelsolin and profilin in human platelets. J. Cell Biol. 105: 833-842, 1987. 19. Madara, J. L., R. Moore, and S. Carlson. Alteration of intestinal tight junction structure and permeability by cytoskeletal contraction. Am. J. Physiol. 253 (Cell Physiol. 22): C854-C861, 1987. 20. Majno, G., and G. E. Palade. Studies on inflammation. I. Effect of histamine and serotonin on vascular permeability: an electron microscopic study. J. Biophys. Biochem. Cytol. 11: 571-605, 1961. 21. Nicolaysen, G. Intravascular concentrations of calcium and magnesium ions and edema formation in isolated lungs. Acta Physiol. Stand. 81: 325-339, 1971. 22. Pasternak, G. R., and R. H. Racusen. Erythrocyte protein 4.1 binds and regulates myosin. Proc. N&l. Acad. Sci USA 86: 97129716, 1989. 23. Rotrosen, D., and J. I. Gallin. Histamine type 1 receptor occupancy increases endothelial cell calcium, reduces F-actin, and promotes albumin diffusion across cultured endothelial monolayers. J. CeZZ BioL. 103: 2379-2387, 1986. 24. Schnittler, H. J., A. Wilke, T. Gress, N. Suttorp, and D. Drenckhahn. Role of actin and myosin in the control of paracellular permeability in pig, rat and human vascular endothelium. J. Physiol. Lond. 431: 379-404, 1990. 25. Shasby, D. M., S. E. Lind, S. S. Shasby, J. C. Goldsmith, and G. W. Hunninghake. Reversible oxidant-induced increases in albumin transfer across cultured endothelium: alterations in cell shape and calcium homeostasis. BZood 65: 605-614, 1985. 26. Shasby, D. M., and S. S. Shasby. Effects of calcium on transendothelial albumin transfer and electrical resistance. J. Appl. PhysioZ. 60: 71-79, 1986. 27. Shasby, D. M., S. S. Shasby, J. M. Sullivan, and M. J. Peach. Role of the endothelial cell cytoskeleton in control of vascular permeability. Circ. Res. 51: 657-661, 1982. 28. Shasby, D. M., M. Yorek, and S. S. Shasby. Exogeneous oxidants initiate hydrolysis of endothelial cell inositol phospholipids. Blood 72: 491-499, 1988. 29. Stossel, T. P. From signal to pseudopod. J. Biol. Chem. 264: 18261-18264, 1989.
Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 10, 2019.
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30. Wysolmerski, R. B., and D. Lagunoff. Inhibition of endothelial cell retraction by ATP depletion. Am. J. Pathol. 132: 28-36, 1988. 31. Wysolmerski, R. B., and D. Lagunoff. Involvement of myosin light-chain kinase in endothelial cell retraction. Proc. NutZ. Acad. Sci. USA 87: 16-20, 1990. 32. Wysolmerski, R. B., D. Lagunoff, and T. Dahms. Ethchlorvynol-induced pulmonary edema in rats. Am. J. PathoZ. 115: 447457, 1984.
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33. Yin, H. L., and T. P. Stossel. Purification and structural properties of gelsolin, a Ca2+ activated regulatory protein of macrophages. J. Biol. Chem. 255: 9490-9493, 1980. 34. Yin, H. L., K. S. Zaner, and T. P. Stosel. Ca2+ control of actin gelation. J. BioZ. Chem. 255: 9494-9500, 1980. 35. Yorek, M. A., J. A. Dunlap, A. A. Spector, and B. H. Ginsberg. Effect of ethanolamine on choline uptake and incorporation into phosphatidylcholine in human Y79 retinoblastoma cells. J. Lipid Res. 27: 1205-1213, 1986.
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MAJNO AND PALADE (20) first reported that adjacent endothelial cells separated from one another when the endothelium was exposed to the edemagenic molecules histamine or serotonin. On the basis of their electron microscopic observations, they suggested that the cells became disconnected along their intercellular junctions. Since then several authors (I, 14, 17, 25-27, 32) have described the development of gaps between adjacent endothelial cells when the endothelium has been exposed to agents that cause edema. Although there are multiple reports of gaps developing between endothelial cells in the setting of edema, the mechanism of the change in endothelial cell shape that causesthe gaps remains poorly understood. Agents that break down actin filaments or that disrupt cell-cell or cell-substrate attachments cause edema and gap formation (21, 26, 27). This suggests that the gaps may result from loss of tethering of cells to each other or to substrate. However, the change in endothelial cell shape caused by histamine is dependent on cell ATP (30). This suggeststhat gap formation in response to histamine is, at least in part, an energy-dependent process. Consistent with this, two groups of investigators found that retraction of the actin cytoskeleton of permeabilized endotheL664
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lial ceils was dependent on activation of myosin lightchain kinase (24, 31). It is conceivable that all of these processes, i.e., release of tethering, changes in actin filaments, and active contraction, contribute to the development of gaps between adjacent endothelial cells exposed to edemagenic molecules. Gelsolin is an actin-binding protein that contributes to changes in cell shape by altering the actin cytoskeleton (6). In the presence of micromolar calcium, gelsolin severs actin filaments and binds to the barbed end of the actin filament, preventing filament elongation. However, gelsolin also promotes actin filament assembly by providing nuclei of filamentous actin (F-actin), which can rapidly assemble into filaments when gelsolin is displaced from actin by the polyphosphoinositides, phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) (6). One of the immediate effects of histamine binding to an H1 receptor on endothelial cells is initiation of inositol phospholipid metabolism and a consequent increase in cell calcium (3, 23). Hence, binding of histamine to an Hi receptor on endothelial cells would likely result in changes in actin-gelsolin interactions, either because of changes in cell calcium or because of changes in polyphosphoinositides, or because of changes in both. In these studies we examined whether histamine activation of signal transduction affected actin-gelsolin interactions in human umbilical vein endothelial (HUVE) cells. METHODS
Materials. Histamine, piperazine-N-N’-bis(2-ethanesulfonic acid) (PIPES), N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES), EGTA, leupeptin, benzamide, aprotinin, phenylmethylsulfonyl fluoride (PMSF), Triton X-100, Na ATP, glucose, and electrolytes were from Sigma Chemical, St. Louis, MO. Monocolonal anti-gelsolin antibodies were conjugated to Sepharose beads as described (18). The specificity of these antibodies has been detailed (4). Tissue culture media and supplies were from GIBCO via the Cancer Center, University of Iowa. Selectamine was from GIBCO. Collagenase was from Worthington Biochemical, Freehold, NJ. Fetal bovine serum (FBS) was from Hyclone, Logan, UT. [““S]methionine(70%)-cysteine(30%) was from ICN, Irvine, CA. [3H]inositol was from New England Nuclear (Boston, MA). Cell culture. HUVE were prepared by collagenase treatment of freshly obtained umbilical veins (3). Harvested cells were plated on 60-mm-diam tissue culture plates (Costar). Cells were cultured in medium 199 (M-199) with 20% FBS, basal medium Eagle vitamins and amino acids, glucose (5 mM), glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 pg/ml). All studies were conducted on primary cu?ures. Such cultures were identified as endothelial by characteristic uniform morphology, uptake of fluorescently labeled acetylated low-density lipo-
0 1992 The
American
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protein, and indirect immunofluorescent staining for factor VIII (3). Cell proteins were labeled with [“5S]methionine by changing the medium 1 day prior to confluence to Selectamine (methionine free) supplemented with 10% FBS and 180 &i/ml [35S]methionine and incubating for 48 h. Labeled cells were then exposed to histamine or vehicle [Hanks’ balanced salt solution (HBSS)] for the indicated times, the labeling media were removed, and the cells were immediately snap frozen on dry icemethanol and then thawed in the lysing buffer as described in Determination of actin-gekolin ratios. A histamine dose of 1 X 10e5 M was used based on our earlier report (3). Cell inositol phospholipids were labeled by adding 4 PCi [3H]inositol to the M-199-20% FBS medium and incubating them for 72 h. After the 72 h, the plates were rinsed with HBSS and then exposed to histamine or vehicle (HBSS) (3). Determination of actin-gelsolin ratios. Cells labeled with [35S]methionine were exposed to histamine or vehicle for the indicated times, the labeling media were removed, and the cells were snap frozen on dry ice-methanol. One milliliter of lysing buffer (120 mM PIPES, 50 mM HEPES, 20 mM EGTA, 4 mM MgClz, 10 mM glucose, 20 kg/ml leupeptin, 156 pg/ml benzamide, 80 pg/ml aprotinin, 1 mM PMSF, and 1.5% Triton X-100, pH 7.2) was added to the plate, and the cells were thawed in the lysing buffer (18). The plates were scraped, and the lysates were centrifuged at 12,000 g for 10 min. Eight hundred microliters of the supernatant solution were added to 30 ~1 of a 50% suspension of anti-gelsolin-Sepharose beads and rotated at 4°C for 2 h, and then the beads were pelleted by centrifugation at 12,000 g for 2 min. The pelleted beads were then washed with 1 ml lysing buffer with 5 mM NaATP, repelleted, and then incubated for 15 min at room temperature in 0.3 M MgC12, 1 mM EGTA, 50 mM tris(hydroxymethyl)aminomethane (Tris) and 100 mM NaCl, pH 7.4, to depolymerize actin filaments associated with the beads (18). After repelleting, the beads were washed with the same solution without the MgClz. Washed beads were boiled in gel sample buffer and loaded onto 5-15% polyacrylamide gels (15). The gels were electrophoresed at 90 V for 15 h, dried, and incubated with X-ray film at -70°C for 60 h. The autoradiograms were developed, and the relative densities of actin and gelsolin were determined by laser densitometry (Molecular Dynamics) . Phospholpid analyses. [3H]inositol (4 mC) in M-199 with 20% FBS was added to each tissue culture plate for 72 h. After the 72 h the medium was removed, and the plates were washed three times with HBSS. HBSS (2 ml) was then added to each plate, and each plate was then exposed to HBSS or histamine (1 x 10m5M). After the indicated time, the medium was removed, the plates were snap frozen on dry ice-methanol, and the cell lipids were extracted according to Bligh and Dyer as previously described (2,3). The chloroform layer was dried under nitrogen, dissolved in chloroform-methanol (2:1), and spotted on preactivated Silica gel 60 plates. The plates were developed in chloroform-methanol-methyl amine-2.4 N HCl (60:35:5:5) (35). Radioactivity in individual lipid fractions was quantitated on a Radiomatics Instruments thin-layer gel scanner (model RS). Standards for phosphatidylinositol, PIP, PIP,, and lysophosphatidylinositol were identified with iodine vapor. In two experiments, cell phospholipids from the chloroform phase of the extraction were spotted onto the same Silica gel plates and separated as above. The inositol phospholipids were identified with standards and scraped into 1.8 ml acid saline for determination of inositol lipid phosphate as described (28). An aliquot of the chloroform phase from the initial Bligh-Dyer extraction was used to determine total cell lipid phosphate. Analysis of Triton-soluble actin. To measure Triton-soluble actin, cytoskeletal proteins were extracted as described by Isaacs and Fulton (10). Briefly, HUVE cells in 60-mm-diam
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tissue culture plates were rinsed free of serum-containing medium and then exposed to HBSS with or without 1 x 10e5 M histamine for the indicated times. Cells were then extracted with 4°C skeleton buffer ( 100 mM KCl, 10 mM PIPES, pH 6.8, 300 mM sucrose, 2 mM MgClz, 1 mM EGTA) with 0.5% Triton X-100, 1 mM PMSF, 100 FM leupeptin, 5 mM benzamide, 5 mM c-aminocaproic acid, and 2 mM iodoacetamide for 10 min. A second extraction with same buffer and protease inhibitors was done at room temperature. These extractions were combined and centrifuged at 100,000 g for 15 min at 4°C. The supernatant was removed and defined as the Triton-soluble skeleton. The cytoskeleton remaining on the tissue culture plate was then extracted with skeleton buffer containing 2% sodium dodecyl sulfate, 0.5% 2-mercaptoethanol, and 100 mM NaCl instead of KCl, sheared through a 26-gauge needle, and the nucleic acids were precipitated by centrifugation at 100,000 g for 15 min. The supernatant from this centrifugation was combined with the precipitate from the Triton extractions and defined as the Triton-insoluble fraction. Aliquots from the Triton-soluble and -insoluble fractions were dissolved in gel sample buffer and chromatographed on 8-15% polyacrylamide gradient gels (15). The gels were stained with Coomassie Blue and dried, and actin content was quantitated by laser densitometry (Molecular Dynamics). Statistical analyses. All data are presented as means f SE. An individual number (n) represents a tissue culture plate. Differences among groups were assessed by analysis of variance with differences between individual groups determined by Tukey highly significant difference test for post hoc comparisons of means. Groups that are referred to as different (greater than or less than) were statistically different at P 5 0.05. RESULTS
Actin-to-gelsolin ratios. After HUVE cells were exposed to histamine, the amount of actin that was immunoprecipitated from the cells with anti-gelsolin antibody decreased relative to the amount of gelsolin that was similarly immunoprecipitated. Figure 1 is an autoradiogram of [35S]methionine-labeled HUVE cell proteins immunoprecipitated with anti-gelsolin antibody coupled to Sepharose beads. Lane 1 is from cells not exposed to
116,250 97,400 66,200
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31,000 Fig. 1. Autoradiogram of [3”S]methionine-labeled proteins immunoprecipitated from human umbilical vein endothelial (HUVE) cells with anti-gelsolin Sepharose. Lane 1, control cells; lane 2, cells exposed to histamine (1 X 10m5M) for 45 s; lane 3, cells exposed to histamine for 90 s.
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histamine, lane 2 is from cells exposed to histamine for 45 s, and lane 3 is from cells exposed to histamine for 90 s. The band at 91 kDa is gelsolin, and the band at 42 kDa is actin. The large band at 56 kDa is believed to be vimentin, based on molecular weight and its abundance in endothelium. After HUVE cells were exposed to histamine, the density of the actin band relative to the gelsolin band rapidly decreased (0.43 t 0.04 at 45 s vs. 0.60 t 0.04 in control). Figure 2 presents a summary of the relative densities of the actin and gelsolin bands immunoprecipitated from [ 35S] methionine-labeled HUVE cells before (Con) and after exposure to histamine for 90 s and 5 min. Triton-soluble actin. The decrease in actin-gelsolin binding observed after exposing HUVE cells to histamine might be expected to enhance the incorporation of actin into the filamentous cytoskeleton (13). To assess this we measured the fraction of actin that was Triton soluble, before and after exposing HUVE cells to histamine. Figure 3 demonstrates that, in HUVE cells exposed to histamine, the fraction of actin that was Triton soluble decreased by 90 s and remained decreased at 5 min. Inositolphospholipids. PIP and PIP, dissociate gelsolin from the barbed end of the actin filament. Because histamine decreased actin-gelsolin binding, it was of interest to determine if histamine increased PIP and PIP, levels in HUVE cells. The mass of total inositol lipid phosphate did not change after HUVE cells were exposed to histamine (Fig. 4). However, the fraction of inositol phospholipid that was present as PIP or PIP2 did increase after HUVE cells were exposed to histamine. Figure 5, A and B, are tracings from the thin-layer gel scanner, demonstrating relative peak areas for PIP2 (peak 1), PIP (peak 2) and PI (peak 3) from [3H]inositol-labeled HUVE cells before (A) and after (B) exposure to histamine. Ninety seconds after HUVE cells were exposed to histamine, PIP had increased as a percent of inositol phospholipid, and by 5 min both PIP and PIP2 had increased as a percent of inositol phospholipid (Fig. 6). Because total inositol phospholipid mass was constant after exposure to histamine, the mass of PIP and PIP2 increased in HUVE cells exposed to histamine. DISCUSSION
Actin is an important constituent of the cortical cytoskeleton of epithelioid cells, where it is linked to spectrin and myosin and to the receptors for cell-cell contact 0.8 .I 5
-I
0.6
.Is 5 v) 0.4 Q) s? s 'g 0.2 a
90 Set 5 Min Fig. 2. Ratios of densities of bands of actin to bands of gelsolin immunoprecipitated from HUVE cells before (Con) and after exposure to histamine for 90 s and 5 min. n = 8 for each time. P 5 0.05 for 90 s and 5 min compared with control.
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Control ’ 90 Set ’ 5 Min ’ Fig. 3. Fraction of actin that was Triton soluble in HUVE cells before and after exposure to histamine for 90 s and 5 min. n = 4 for each time. P 5 0.05 for 90 s and 5 min compared with control.
Con
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Fig. 4. Inositol lipid phosphate in HUVE cells before and after exposure to histamine for 90 s and 5 min. n = 6 for each time. There were no differences in inositol lipid phosphate at different times.
and cell-substrate contact (18, 21). Molecules such as the cytochalasins, which alter the physiochemical state of actin, alter epitheliod cell shape and the integrity of epithelial and endothelial barriers (19, 27). Edemagenic molecules such as histamine alter endothelial cell shape, and it is likely that histamine-mediated changes in endothelial cell shape involve changes in actin in endothelial cells. Gelsolin is an actin-binding protein that can shorten actin filaments and block filament assembly when it binds to the barbed end of actin filaments. Dissociation of gelsolin from the barbed end of actin filaments can also promote rapid filament assembly by providing a large number of oligomers with a free barbed end (6, 29). The binding of gelsolin to actin is stimulated by micromolar calcium and inhibited by PIP and PIP2 (33, 34). When macrophages and platelets are activated, gelsolin approaches the plasma membrane, where it could be exposed to high local concentrations of these polyphosphoinositides. Because binding of histamine to the H1 receptor on endothelial cells activates inositol phospholipid metabolism and increases calcium, it was likely that histamine would alter actin-gelsolin interactions in endothelial cells (3). We found that within 45 s of adding histamine to [35S]methionine-labeled HUVE cells, the relative densities of the actin/gelsolin bands decreased from control levels of binding is 0.6 to -0.4. This decrease in actin-gelsolin similar to the decrease in actin-gelsolin binding found by Dadabay et al. (7) in A 431 cells exposed to epithelial growth factor (EGF). Dissociation of actin from gelsolin would be expected to
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100 % a
80 60
z
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Con
90 Set
5 Min
10 8 6 4 2 0 Con Fig. 6. Distribution labeled HUVE cells mine (1 X low5 M); min compared with control.
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Fig. 5. A: tracing of thin-layer chromatogram from [3H]inositol-labeled HUVE cells before exposure to histamine. Peak 1 represents phosphatidylinositol 4,5bisphosphate (PIP2), peak 2 represents phosphatidylinositol 4-phosphate (PIP), and peak 3 represents phosphatidylinositol. B: tracing of similar thin-layer chromatogram from HUVE cells 5 min after exposure to histamine (1 X 10e5 M).
increase incorporation of actin into microfilaments. Cell actin is frequently functionally divided into the Tritonsoluble (nonfilamentous) and -insoluble (filamentous) fractions for purposes of estimating how much of the actin is incorporated into microfilaments (10). We found that the fraction of actin that was Triton insoluble increased after histamine exposure, consistent with the decrease in actin-gelsolin binding. This increase in F-actin is consistent with the findings of Dadabay et al. (7), who found that EGF increased F-actin in A 431 cells, and with those of Eberle et al. (8), who found that N-formyl-MetLeu-Phe increased actin polymerization in neutrophils. Because histamine rapidly increases endothelial cell calcium, it was surprising that the initial response was not an increase in actin-gelsolin association. Histamine increases endothelial cell calcium to -500 nM, whereas the affinity constant of gelsolin for calcium is 920 nM (6, 33, 34). Hence, histamine may not increase endothelial cell calcium enough to increase actin-gelsolin binding. However, since our measurements represent the net average actin-gelsolin binding in the whole cell, our data do
90 Set
of counts in inositol before and 90 s and n = 9 for each time. control. P 5 0.05 for
5 Min
phospholipids in [3H]inositol5 min after exposure to histaP 5 0.05 for PIP at 90 s and 5 PIP2 at 5 min compared with
not exclude the possibility that actin-gelsolin binding increases within specific cellular regions. There have been two independent reports that calciummediated changes in the shape of the actin cytoskeleton of detergent-permeabilized endothelial cells is dependent on activation of phosphorylation of the light chain of myosin (24, 31). Phosphorylation of the light chain of myosin is important for the maintenance and assembly of actin microfilaments in nonmuscle cells (16). Both phosphorylation of the myosin light chain and dissociation of actin from gelsolin would enhance microfilament assembly. Hence, the effects of histamine on actin-gelsolin would complement effects of histamine on calcium-initiated myosin light chain phophorylation in endothelium and promote the observed increase in F-actin. In addition to increasing endothelial cell calcium, we found that histamine increased endothelial cell PIP and PIP2. In vitro, increased concentrations of PIP or PIP2 decrease actin-gelsolin binding and promote actin filament assembly (1 l- 13). In activated macropha .ges and platelets, actin- #gelsolin complexes associate with the cell membrane where they would be exposed to high local concentrations of PIP and PIP2 (9). Based on the these observations, the observed histamine-initiated increases in PIP and PIP2 may be causally related to the detected decreases in actin-gelsolin binding. Whereas Dadabay et al. (7) found that EGF decreased actin-gelsolin binding in A 431 cells, their data did not support the hypothesis that changes in PIP and PIP,, might cause the decreased binding. However, there are some important differences between A 431 and HUVE cells and their responses to the respective agonists. EGF increased PIP in A 431 cells but, unlike HUVE cells stimulated with histamine, EGF did not increase, but actually decreased PIP2 in A 431 cells. Hence, there is a difference in inositol metabolism between the cells after
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stimulation with the respective agonists. Additionally, cholera toxin pretreatment prevented the EGF-stimulated increase in PIP but did not prevent membrane ruffling or the decrease in actin-gelsolin binding in A 431 cells (7). In contrast, pretreatment of HUVE cells with agents which increase CAMP, prevents cell retraction and myosin light chain phosphorylation, both of which imply major effects of CAMP on F-actin (Refs. 3 and 15 and A. Moy and D. M. Shasby, unpublished observations). Hence, in HUVE cells it would not be possible to independently interpret effects of CAMP on the actin cytoskeleton and on inositol phospholipid metabolism. Eberle et al. (8) observed that increased F-actin in N-formyl-Met-Leu-Phe-activated neutrophils was associated with an increase in phosphatidylinositol trisphosphate (PIPS) in the neutrophils. We did not measure PIP3 in HUVE cells exposed to histamine and therefore cannot comment on the relevance of Eberle’s observations to ours. However, in the neutrophils, PIP2 decreased after activation, in contrast to the increase in PIP2 in HUVE cells exposed to histamine. Whether some of the increase in PIP2 measured in HUVE cells exposed to histamine represents comigration of PIP2 and PIP3 is not possible for us to determine at this time. In summary, histamine changes endothelial cell shape, and the change in cell shape likely involves changes in the actin cytoskeleton. Gelsolin is an actin-binding protein with the capacity to profoundly alter the actin cytoskeleton in response to calcium or in response to polyphosphoinositides. In HUVE cells activated with histamine, gelsolin interactions with actin are better predicted by changes in HUVE cell polyphosphoinositides than by the increases in cell calcium. This research was completed during D. M. Shasby’s tenure as a Clinical Investigator of the Veteran’s Administration. The work was also supported by National Heart, Lung, and Blood Institute Grant HL-33540. Address for reprint requests: D. M. Shasby, Dept. of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA, 52242. Received
7 May
1991; accepted
in final
form
10 August
1992.
REFERENCES 1. Albertine, K. H., J. P. Weiner-Kronish, K. Koike, and N. C. Staub. Quantification of damage to lung microvessels in anesthetized sheep. J. Appl. Physiol. 57: 1360-1368, 1984. 2. Bligh, E. G., and W. J. Dyer. A rapid method of total lpid extraction and purification. Can. J. Biochem. 37: 911-917, 1959. 3. Carson, M. R., S. S. Shasby, and D. M. Shasby. Histamine and inositol phosphate accumulation in endothelium: CAMP and a G protein. Am. J. Physiol. 257 (Lung Cell. Mol. Physiol. 1): L259L264, 1989. 4. Chaponnier, C., P. A. Janmey, and H. L. Yin. The actin filament-severing domain of plasma gelsolin. J. CeZZ Biol. 103: 1473-1481, 1986. 5. Chaponnier, C., H. L. Yin, and T. P. Stossel. Reversibility of gelsolin. Actin interaction in macrophages: evidence for Ca2+ dependent and Ca2+ independent pathways. J. Exp. Med. 165: 97106, 1987. 6. Cunningham, C. C., T. P. Stossel, and D. J. Kwiatowski. Enhanced motility in NIH 3T3 fibroblasts that overexpress gelsolin. Science Wash. DC 251: 1233-1236, 1991. 7. Dadaby C. Y., E. Patton, J. A. Cooper, and L. J. Pike. Lack of correlation bewteen changes in polyphosinositide levels and actin/gelsolin complexes in A 431 cells treated with epidermal prowth factor. J. CeZZ BioZ. 112: 1151-1156. 1991.
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8. Eberle, M., A. E. Traynor-Kaplan, L. A. Sklar, and J. Norgauer. Is there a relationship between phosphatidykinositol trisphosphate and F-actin polymerization in human neutrophils? J. BioZ. Chem. 265: 16725-16728, 1990. 9. Hartwig, J. H., K. A. Chambers, and T. P. Stossel. Association of gelsolin with actin filaments and cell membranes of macrophages and platelets. J. CeZZ BioZ. 108: 467-479, 1989. 10. Isaacs, W. B., and A. B. Fulton. Cotranslational assembly of myosin heavy chain in developing cultured skeletal muscle. Proc. Natl. Acad. Sci. USA 84: 6174-6178, 1987. 11. Janmey, P. A., K. Iida, H. L. Yin, and T. P. Stossel. Polyphosphoinositide micelles and polyphosphoinositide-containing vesicles dissociate endogenous gelsolin-actin complexes and promote actin assembly from the fast-growing end of actin filaments blocked by gelsolin. J. BioZ. Chem. 262: 12228-12236, 1987. 12. Janmey, P. A., and T. P. Stossel. Modulation of gelsolin function by phosphatidylinositol 4,5-bisphosphate. Nature Lond. 325: 362-364, 1987. 13. Janmey, P. A., and T. P. Stossel. Gelsolin-polyphosphoinositide interaction. J. Biol. Chem. 264: 4825-4831, 1989. 14. Kadish, J. L., C. E. Butterfield, and J. Folkman. The effect of fibrin on cultured vascualr endothelial cells. Tissue CeZZ 11: 99-108, 1979. 15. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature Lond. 227: 680685, 1970. 16. Lamb, N. J. C., A. Fernandez, M. A. Conti, R. Adelstein, D. B. Glass, W. J. Welch, and J. R. Feramisco. Regulation of actin microfilament integrity in living nonmuscle cells by the CAMP-dependent protein kinase and the myosin light chain kinase. J. CeZZ BioZ. 106: 1955-1971, 1988. 17. Laposata M., D. K. Dovnarsky, and H. S. Shin. Thrombininduced gap formation in confluent endothelial cell monolayers in vitro. BZood 62: 549-556, 1983. 18. Lind, S. E., P. A. Janmey, C. Chaponnier, T.-J. Herbert and T. P. Stossel. Reversible binding of actin to gelsolin and profilin in human platelets. J. Cell Biol. 105: 833-842, 1987. 19. Madara, J. L., R. Moore, and S. Carlson. Alteration of intestinal tight junction structure and permeability by cytoskeletal contraction. Am. J. Physiol. 253 (Cell Physiol. 22): C854-C861, 1987. 20. Majno, G., and G. E. Palade. Studies on inflammation. I. Effect of histamine and serotonin on vascular permeability: an electron microscopic study. J. Biophys. Biochem. Cytol. 11: 571-605, 1961. 21. Nicolaysen, G. Intravascular concentrations of calcium and magnesium ions and edema formation in isolated lungs. Acta Physiol. Stand. 81: 325-339, 1971. 22. Pasternak, G. R., and R. H. Racusen. Erythrocyte protein 4.1 binds and regulates myosin. Proc. N&l. Acad. Sci USA 86: 97129716, 1989. 23. Rotrosen, D., and J. I. Gallin. Histamine type 1 receptor occupancy increases endothelial cell calcium, reduces F-actin, and promotes albumin diffusion across cultured endothelial monolayers. J. CeZZ BioL. 103: 2379-2387, 1986. 24. Schnittler, H. J., A. Wilke, T. Gress, N. Suttorp, and D. Drenckhahn. Role of actin and myosin in the control of paracellular permeability in pig, rat and human vascular endothelium. J. Physiol. Lond. 431: 379-404, 1990. 25. Shasby, D. M., S. E. Lind, S. S. Shasby, J. C. Goldsmith, and G. W. Hunninghake. Reversible oxidant-induced increases in albumin transfer across cultured endothelium: alterations in cell shape and calcium homeostasis. BZood 65: 605-614, 1985. 26. Shasby, D. M., and S. S. Shasby. Effects of calcium on transendothelial albumin transfer and electrical resistance. J. Appl. PhysioZ. 60: 71-79, 1986. 27. Shasby, D. M., S. S. Shasby, J. M. Sullivan, and M. J. Peach. Role of the endothelial cell cytoskeleton in control of vascular permeability. Circ. Res. 51: 657-661, 1982. 28. Shasby, D. M., M. Yorek, and S. S. Shasby. Exogeneous oxidants initiate hydrolysis of endothelial cell inositol phospholipids. Blood 72: 491-499, 1988. 29. Stossel, T. P. From signal to pseudopod. J. Biol. Chem. 264: 18261-18264, 1989.
Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 10, 2019.
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30. Wysolmerski, R. B., and D. Lagunoff. Inhibition of endothelial cell retraction by ATP depletion. Am. J. Pathol. 132: 28-36, 1988. 31. Wysolmerski, R. B., and D. Lagunoff. Involvement of myosin light-chain kinase in endothelial cell retraction. Proc. NutZ. Acad. Sci. USA 87: 16-20, 1990. 32. Wysolmerski, R. B., D. Lagunoff, and T. Dahms. Ethchlorvynol-induced pulmonary edema in rats. Am. J. PathoZ. 115: 447457, 1984.
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33. Yin, H. L., and T. P. Stossel. Purification and structural properties of gelsolin, a Ca2+ activated regulatory protein of macrophages. J. Biol. Chem. 255: 9490-9493, 1980. 34. Yin, H. L., K. S. Zaner, and T. P. Stosel. Ca2+ control of actin gelation. J. BioZ. Chem. 255: 9494-9500, 1980. 35. Yorek, M. A., J. A. Dunlap, A. A. Spector, and B. H. Ginsberg. Effect of ethanolamine on choline uptake and incorporation into phosphatidylcholine in human Y79 retinoblastoma cells. J. Lipid Res. 27: 1205-1213, 1986.
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