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(Circulation Research. 2000;86:456.)
© 2000 American Heart Association, Inc.

Cellular Biology

_vß₃ Integrin Induces Tyrosine Phosphorylation–Dependent Ca²⁺ Influx in Pulmonary Endothelial Cells

Sunita Bhattacharya, Xiaoyou Ying, Chenzhong Fu, Rashmi Patel, Wolfgang Kuebler, Steven Greenberg, Jahar Bhattacharya

From the Departments of Pediatrics (S.B.) and Medicine (X.Y., C.F., R.P., W.K., J.B.), and St Luke’s–Roosevelt Hospital Center, and the Departments of Pediatrics (S.B.), Physiology & Cellular Biophysics (X.Y., W.K., J.B.), Medicine (J.B., S.G.), and Pharmacology (S.G.), College of Physicians and Surgeons, Columbia University, New York, NY.

Correspondence to Dr Sunita Bhattacharya, St Luke’s–Roosevelt Hospital Center, 1000 10th Ave, New York, NY 10019. E-mail Sb80{at}columbia.edu

	Abstract

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Abstract—The endothelial _vß₃ integrin occurs luminally, where its ligation by soluble agents may induce inflammatory signaling. We tested this hypothesis in bovine pulmonary artery endothelial cell monolayers with the use of vitronectin and cross-linking antibodies to ligate and aggregate the integrin. We quantified the endothelial cytosolic Ca²⁺ concentration ([Ca²⁺]_i) according to the Fura 2 ratio imaging method in single cells of confluent monolayers. At baseline, endothelial [Ca²⁺]_i levels remained steady at 86 nmol/L for >20 minutes. Cross-linking of the _vß₃ integrin through the sequential exposure of monolayers to anti-_vß₃ monoclonal antibody LM609 and secondary IgG resulted in a [Ca²⁺]_i increase of 100% above baseline. This increase commenced in <0.5 minute, peaked in <2 minutes, and decayed to baseline in 5 minutes. Similar responses occurred after the addition of vitronectin (400 µg/mL). In contrast, external Ca²⁺ depletion blunted the cross-linking–induced [Ca²⁺]_i increase by 60%, a response that was completely inhibited when the monolayers were also pretreated with thapsigargin. Thus, the [Ca²⁺]_i increase was attributable in part to the release of Ca²⁺ from endosomal stores but mostly to Ca²⁺ influx across the plasma membrane. Induced aggregation of the _vß₃ integrin enhanced tyrosine phosphorylation of phospholipase C-1 and increased the accumulation of inositol-1,4,5-trisphosphate. Genistein, a broad-spectrum tyrosine kinase inhibitor, abrogated both of these effects, as well as the _vß₃-induced [Ca²⁺]_i increases. We conclude that aggregation of the endothelial _vß₃ integrin induces a rapid tyrosine phosphorylation–dependent increase in [Ca²⁺]_i. This response may subserve the inflammatory role of _vß₃ integrin in blood vessels.

Key Words: integrins • endothelium • cells • vitronectin • phospholipases

	Introduction

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The endothelial _vß₃ integrin is important in angiogenesis, vasculogenesis, and vascular cell survival.^{1 2} These are matrix-dependent functions that probably in large part involve integrins expressed on the matrix-facing aspect of the endothelial cell. However, the endothelial _vß₃ integrin also exists on the luminal, blood-facing aspect,³ where it is likely to function as a receptor for blood-borne ligands. Our finding that the luminal _vß₃ integrin increases lung capillary permeability⁴ indicates that the luminal integrin may subserve inflammatory endothelial responses.

Rapid mobilization of endothelial Ca²⁺ is often characteristic of inflammatory processes. Increases in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) trigger vascular responses such as an increase in capillary permeability,⁵ the secretion of inflammatory cytokines,⁶ and the induction of gene transcription.⁷ However, _vß₃-induced [Ca²⁺]_i responses have not been determined in stable endothelial cells.⁸ In endothelial cells allowed to spread on immobilized _vß₃ ligands, [Ca²⁺]_i increases occur gradually and reach a peak at 30 minutes⁸ These slow [Ca²⁺]_i increases do not account for rapid endothelial responses such as barrier deterioration that occur in <1 minute.⁴ Soluble _vß₃ ligands decrease [Ca²⁺]_i in osteoclasts⁹ and myocytes,¹⁰ a response that if true for endothelial cells, may argue in favor of a barrier-protective, not a barrier-deteriorating, effect.

Receptor-mediated endothelial [Ca²⁺]_i increases may result from a sequence in which phospholipase C- (PLC-) activation leads to the release of inositol-1,4,5-triphosphate (InsP₃), store release of Ca²⁺, and entry of external Ca²⁺.¹¹ These well reported mechanisms apply to several receptors that activate PLC- through tyrosine phosphorylation.¹² Although the _vß₃ integrin may fall in this category,¹² mechanisms remain confusing because protein tyrosine phosphorylation may itself be Ca²⁺ enhanced.^{13 14} However, it is also possible that [Ca²⁺]_i elevation inhibits the phosphorylation.^{15 16} We considered these possibilities in the context of the _vß₃ integrin.

Multivalent vitronectin aggregates the _vß₃ integrin and enhances protein tyrosine phosphorylation.¹⁷ Complement activation increases plasma levels of the vitronectin-containing complement complexes.¹⁸ Such complexes, as well as other vitronectin-binding inflammatory factors, such as bacteria, viruses,^{19 20 21} and the multivalent thrombin-antithrombin complex,²² may ligate the luminal _vß₃ integrin during inflammatory and thrombotic states. Ensuing _vß₃ integrin–mediated enhanced protein tyrosine phosphorylation may then initiate inflammatory responses.

In the present study, we used pulmonary endothelial monolayers to model responses relevant to our previously reported findings in lung capillaries.⁴ Our main aim was to determine whether _vß₃ ligation sufficiently aggregates the integrin to rapidly increase endothelial [Ca²⁺]_i. Our strategy was to aggregate the integrin with the use of cross-linking antibodies or multimeric vitronectin. Our findings indicate that both caused tyrosine phosphorylation–induced [Ca²⁺]_i increases that unexpectedly initiated at the cell periphery. We discuss the implications of these findings in relation to the permeability-enhancing effect of the integrin in capillaries.

	Materials and Methods

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Cells, Reagents, and Antibodies
Bovine pulmonary artery endothelial cells (BPAECs) (American Type Culture Collection) were grown to confluence in DMEM, and immunofluorescent staining for factor VIII antigen was confirmed. EGTA, genistein, and thapsigargin were purchased from Sigma Chemical Co. MAPTAM (1,2-bis-5-methyl-amino-phenoxylethane-N,N,N'-tetra-acetoxymethyl acetate) was purchased from Calbiochem. Secondary antibody (Ab) and donkey anti-mouse IgG were purchased from Jackson ImmunoResearch, Inc. Polyclonal antiserum against vitronectin and the anti-_vß₅ Ab PIF6 were obtained from Chemicon. Affinity-purified polyclonal rabbit IgG against phosphotyrosine was obtained from ICN Biomedicals. Monoclonal Ab (mAb) against PLC-1 was purchased from Transduction Laboratories. Protein A/protein G-agarose was obtained from Santa Cruz Biotechnology. Fluorolink-Ab Cy3 labeling kit was purchased from Amersham. The InsP₃ assay kit was obtained from New England Nuclear. mAb LM609 against the _vß₃ integrin was generously provided by D. Cheresh (Scripps Clinic and Research Foundation, La Jolla, Calif). Multimeric vitronectin was purified from human plasma as described previously.¹⁷

Cross-Linking Protocols
The _vß₃ integrin was cross-linked through the exposure of confluent BPAEC monolayers first to LM609 (200 µg/mL, 30 minutes, 4°C) and then to donkey anti-mouse IgG (30 µg/mL, 5 minutes, 37°C). For Ca²⁺-free conditions, the secondary IgG was added in Ca²⁺-free buffer containing 0.5 mmol/L EGTA. For the immunofluorescent detection of _vß₃ aggregation, BPAECs were lightly fixed (3.7% paraformaldehyde, 20 minutes, 22°C) and permeabilized (0.5% Triton X-100, 2 minutes) according to Lawson et al.²³ Then, _vß₃ was cross-linked with Cy3-conjugated LM609 as the primary Ab. Immunofluorescence was detected with confocal (Insight+; Meridian Instrument Co) and conventional (Olympus LH50A) fluorescence microscopy.

Ca²⁺ Imaging of Single Endothelial Cells
Our methods for Ca²⁺ imaging according to the Fura 2 ratio method were described previously.²⁴ BPAEC monolayers were Fura 2 loaded through the addition of Fura 2-AM (5 µmol/L, 30 minutes, 20°C) and then maintained at 37°C during digital imaging. [Ca²⁺]_i was determined in a 2-µm² window placed over 340/380 ratio images of single cells, based on appropriate calibrations and a Fura 2/Ca²⁺ K_D value of 224 nmol/L.²⁵

Immunoblotting and Immunoprecipitation
Cells were lysed in ice-cold lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris base, 2 mmol/L EDTA, 50 mmol/L NaF, 0.1% SDS, 1% NP-40, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, and 1 mmol/L sodium orthovanadate, the phosphatase inhibitor, pH 7.5). Lysates were cleared through centrifugation (14 000 rpm, 15 minutes), and protein concentrations were determined according to the DC Protein Assay (Bio-Rad). Anti-phosphotyrosine immunoblotting was performed as described previously.¹⁷ Cell lysates containing equal amounts of protein were electrophoresed onto 10% SDS-polyacrylamide gels under reducing conditions. After electrophoretic transfer to nitrocellulose, phosphotyrosyl-containing proteins were detected with affinity-purified anti-phosphotyrosine IgG that was previously derivatized with sulfosuccinimidylbiotin, followed by the addition of streptavidin-horseradish peroxidase. Blots were developed with the use of enhanced chemiluminescence. Immunoprecipitation was performed as described previously.¹⁷

Statistical Analysis
All values are given as mean±SEM. Differences between groups were tested with the paired t test for 2 groups and the Newman-Keuls test for >2 groups. Statistical significance was accepted at P<0.05.

	Results

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Immunofluorescence Studies of Clustered _vß₃ in BPAEC Monolayers
To determine [Ca²⁺]_i responses to _vß₃ clustering, we used cross-linking Abs as well as vitronectin. The binding affinity of vitronectin to the _vß₃ integrin is Ca²⁺ dependent.²⁶ The advantage of cross-linking is that the binding affinity of the anti-_vß₃ mAb LM609 is both _vß₃ specific and Ca²⁺ independent.²⁷ Accordingly, cross-linking permitted determinations of aggregation responses under Ca²⁺-depleted conditions.

Sites of _vß₃ aggregation were determined with both conventional and confocal microscopy. The addition of mAb LM609 alone resulted in diffuse fluorescence on the cell surface (Figures 1A and 1C). However, cross-linking of the mAb resulted in the formation of fluorescent clumps, signifying _vß₃ aggregation in all viewed cells. The clumps were located largely at the cell periphery (Figure 1B) and were viewed best at the superficial confocal levels (Figure 1D). Similar clustering patterns occurred with the addition of vitronectin (400 µg/mL) and with cross-linking under external Ca²⁺-free conditions, indicating that the aggregation was not Ca²⁺ dependent.

View larger version (146K): [in this window] [in a new window]   Figure 1. mAb LM609–induced aggregation of vß3. BPAEC monolayers fluorescently labeled with Cy3 conjugated with mAb LM609 were viewed after addition of buffer (A and C) or cross-linking IgG (B and D). Images by conventional (A and B) and confocal (C and D) microscopy of apical surfaces of single cells indicate aggregates (arrows). Replicated 3 times for each condition. LM609 indicates anti-vß3 Ab; 2° Ab, secondary Ab (donkey anti-mouse IgG).

[Ca²⁺]_i Responses to Clustering _vß₃
Figure 2 shows single cell images, exemplifying [Ca²⁺]_i response patterns. Cross-linking (Figure 2, top) or the addition of multimeric vitronectin (not shown) caused [Ca²⁺]_i increases that initiated at the cell periphery and then spread centripetally. In contrast, histamine-induced [Ca²⁺]_i increases occurred more globally and usually initiated at the cell center and then spread outward (Figure 2, bottom).

View larger version (60K): [in this window] [in a new window]   Figure 2. vß3 aggregation causes peripherally initiated endothelial [Ca2+]i transients. LM609 indicates anti-vß3 Ab; 2° Ab, secondary Ab (donkey anti-mouse IgG). High-magnification fluorescence microscopy of single cells in Fura 2–loaded BPAEC monolayers. Pseudocolor images depict time-dependent [Ca2+]i distributions. Top, Response of a single cell to vß3 cross-linking. Bottom, Response of a single cell from a different monolayer exposed to 1 µmol/L histamine. [Ca2+]i given in nmol/L. A indicates baseline; B, 30 seconds; C, 60 seconds; D, 90 seconds.

At baseline, [Ca²⁺]_i determinations in 19 monolayers (10 cells per monolayer) averaged 86±14 nmol/L. Representative [Ca²⁺]_i tracings from single cells are shown in Figure 3. Both cross-linking and vitronectin increased [Ca²⁺]_i (Figures 3A and 3B) in 80±5% of cells (19 monolayers). On average, the increase commenced in 0.5±0.2 minute, peaked at 1.5±0.3 minutes, and recovered to within 10% of baseline by 5±0.4 minutes (n=8). [Ca²⁺]_i oscillations were usually evident in the recovery period. Cross-linking under control conditions caused a peak increase in [Ca²⁺]_i by 85±13 nmol/L (n=12; P<0.01). In contrast, under Ca²⁺-free conditions, cross-linking increased [Ca²⁺]_i by only 27±5 nmol/L, indicating marked blunting of the response (n=7; P<0.01). The vitronectin-induced [Ca²⁺]_i increase could be blocked by preincubation of the monolayer with mAb LM609 (Figure 3C; P<0.01, n=4).

View larger version (24K): [in this window] [in a new window]   Figure 3. Single-cell [Ca2+]i responses in BPAEC monolayers. LM609 indicates anti-vß3 Ab; MAP, Ca2+ chelator MAPTAM (200 µmol/L); IgG1, nonspecific Ab isotype matched with LM609; PIF6, anti-vß5 Ab. Boxed text indicates agent with which monolayer was incubated for 30 minutes before addition of secondary Ab for cross-linking LM609 (2y), thapsigargin (TH, 2 µmol/L), or vitronectin (VN, 400 µg/mL). Ext Ca2+(-) indicates secondary Ab was in Ca2+-depleted solution containing 0.5 mmol/L EGTA. Each experiment was replicated 5 times.

Although cross-linking under Ca²⁺-free conditions caused a blunted [Ca²⁺]_i increase, the subsequent addition of thapsigargin, the endosomal Ca²⁺-ATPase inhibitor, markedly increased [Ca²⁺]_i (Figure 3D) (P<0.05, n=4).²⁸ Because thapsigargin induces Ca²⁺ release from endosomal stores, this result indicates that external Ca²⁺-free conditions did not cause store depletion. However, when we first used thapsigargin to cause store depletion and then cross-linked monolayers in Ca²⁺-free conditions, all [Ca²⁺]_i increases were blocked (Figure 3E; P<0.05, n=4). We interpret that clustering of the _vß₃ integrin induced [Ca²⁺]_i transients arising from both the release of intracellular Ca²⁺ stores and the influx of external calcium across the plasma membrane. Also shown are data from several experiments carried out to validate our procedures (n=4 each). Thus, the intracellular Ca²⁺-chelator MAPTAM (Figure 3F, 200 µmol/L) completely blocked cross-linking–induced [Ca²⁺]_i increases. No [Ca²⁺]_i responses resulted after the addition of secondary Ab to either untreated monolayers (Figure 3G) or monolayers preincubated with an IgG₁ isotype matched with LM609 (Figure 3H) or an mAb against the _vß₅ integrin (Figure 3I). Therefore, the cross-linking responses were _vß₃ specific. Moreover, the responses to vitronectin were concentration dependent (Figure 4).

View larger version (15K): [in this window] [in a new window]   Figure 4. Group responses to vitronectin (VN). Points are peak [Ca2+]i responses (mean±SEM) in 4 BPAEC monolayers. *P<0.01 compared with baseline.

Protein Tyrosine Phosphorylation and [Ca²⁺]_i Regulation in BPAECs
In affirmation of our previous findings,¹⁷ cross-linking _vß₃ consistently enhanced tyrosine phosphorylation of several proteins, especially those corresponding to a kDa value of 125, 68, 62, 52, 48, and 34 (Figure 5, lane 3). The addition of mAb LM609 alone had no effect (not shown). External Ca²⁺ removal, the addition of thapsigargin, and the addition of MAPTAM, the intracellular Ca²⁺ chelator, had no effects on the cross-linking–induced tyrosine phosphorylation of the 6 bands of interest (compare lanes 3, 5, and 7 in Figure 5). Hence, Ca²⁺ depletion did not affect tyrosine kinase activation relevant to these proteins.

View larger version (70K): [in this window] [in a new window]   Figure 5. Protein tyrosine phosphorylation induced by vß3 aggregation. Anti-phosphotyrosine immunoblots are shown for endothelial lysates from BPAEC monolayers. Control mAb indicates nonspecific Ab isotype matched with LM609; mAb LM609, anti-vß3 mAb; 2° Ab, secondary Ab (donkey anti-mouse IgG); Ext Ca2+, extracellular Ca2+ present (+) or depleted (-); Thapsi, thapsigargin (2 µmol/L); MAPTAM, intracellular Ca2+ chelator (200 µmol/L). Molecular weight markers (in kDa) shown on left. Data are representative of 4 separate experiments.

When we combined external Ca²⁺ removal with either thapsigargin or MAPTAM, tyrosine phosphorylation decreased on the band at 125 kDa. External Ca²⁺ removal alone or in combination with thapsigargin or MAPTAM reduced tyrosine phosphorylation on several bands of less than 46 kDa (lanes 4, 6, and 8 in Figure 5). Hence, _vß₃-induced tyrosine phosphorylation was Ca²⁺ independent for some, but not all, endothelial proteins.¹³

We determined whether clustering the _vß₃ integrin on BPAECs induced enhanced tyrosine phosphorylation of PLC-1, which underlies InsP3-induced Ca²⁺ fluxes evoked by other tyrosine kinase–mobilizing receptors.¹² Immunoprecipitation experiments indicated that cross-linking _vß₃ enhanced tyrosine phosphorylation of PLC-1 and that the effect was blocked by the tyrosine kinase inhibitor genistein (100 µmol/L) (Figure 6A).²⁹ We also determined that the addition of multimeric vitronectin or the clustering of cell surface _vß₃ led to modest but significant increases in InsP₃ accumulation that were also abrogated by the addition of genistein (Figure 6B). Genistein also completely inhibited the [Ca²⁺]_i increase in response to vitronectin (Figure 6C) as well as to cross-linking (not shown). Hence, the tyrosine kinase inhibitor blocked all 3 _vß₃-induced effects: tyrosine phosphorylation of PLC-1, increased InsP₃ production, and increased [Ca²⁺]_i.

View larger version (20K): [in this window] [in a new window]   Figure 6. Inhibition of vß3-induced responses in BPAECs. Control mAb indicates nonspecific Ab isotype matched with LM609; mAb LM609, anti-vß3 mAb; 2° Ab, secondary Ab (donkey anti-mouse IgG); GN, genistein (100 µmol/L). A, BPAEC monolayers were cross-linked using mAb LM609 or isotype-matched mAb (control mAb) in presence or absence of genistein (37°C, 15 minutes, lane 3) as indicated. PLC-1 was immunoprecipitated (IP) from lysates and subjected to SDS-PAGE, transfer, and immunoblotting (IB) with either anti–PLC-1 (top row) or anti-phosphotyrosine (PY) (bottom row) Abs. Data represent 3 separate experiments. B and C, InsP3 and [Ca2+]i determined as indicated in BPAEC monolayers exposed to vitronectin (VN, 400 µg/mL) or after cross-linking (CL) of vß3 integrin in absence or presence of genistein. Data are mean±SE. *P<0.01 compared with bar on left (n=number of monolayers).

To test for nonspecific inhibition of Ca²⁺ mobilization mechanisms, we determined that genistein did not inhibit histamine-induced [Ca²⁺]_i increases, although it attenuated the effect. Thus, compared with control experiments in which histamine (10 µmol/L) increased [Ca²⁺]_i by 80±12% above baseline (70±4 nmol/L) (P<0.05, n=3), the response was attenuated in genistein-treated cells in which the increase was 30±7% (P<0.05, n=3).

	Discussion

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We reported previously that ligation of the lung endothelial _vß₃ integrin causes rapid enhancement of protein tyrosine phosphorylation.¹⁷ Here we show that an important consequence of this phosphorylation is the triggering of rapid [Ca²⁺]_i increases. A comparison of our previous and present data indicates that the induced tyrosine phosphorylation and the [Ca²⁺]_i increases occurred with a similar time course. Importantly, PLC-1 was tyrosine phosphorylated and possibly activated as indicated by the increase in InsP₃, a product of PLC-1–induced hydrolysis on inositol bisphosphate.¹² The PLC-1 phosphorylation, the InsP₃ increases, and the [Ca²⁺]_i increases were completely inhibited with genistein. By contrast, histamine-induced [Ca²⁺]_i increases occurred in the presence of genistein, indicating that genistein caused no nonspecific inhibition of Ca²⁺ mobilization. We conclude from these findings that interaction of the endothelial _vß₃ integrin with soluble ligands induced tyrosine phosphorylation as the primary mechanism for increasing endothelial [Ca²⁺]_i.

The [Ca²⁺]_i increases were attributable in large part to Ca²⁺ entry, because they were 70% abrogated when external Ca²⁺ was depleted. However, a significant intracellular component was present, because the residual 30% of the response was blocked by prior treatment with thapsigargin, which depletes endosomal Ca²⁺ stores through inhibition of the endosomal Ca²⁺-ATPase pump.²⁸ Therefore, this intracellular component was likely due to Ca²⁺ release from thapsigargin-sensitive ER stores. InsP₃ ligates endosomal receptors to cause store release of Ca²⁺. Further [Ca²⁺]_i increases likely result from the Ca²⁺-induced Ca²⁺-release mechanisms,¹¹ as well as by an inadequately understood mechanism via which store depletion causes entry of external Ca²⁺.³⁰ As summarized in Figure 7, the present InsP₃ increase is likely to have induced these [Ca²⁺]_i-increasing mechanisms.

View larger version (16K): [in this window] [in a new window]   Figure 7. Signaling pathways for [Ca2+]i regulation by vß3 integrin. ER indicates endoplasmic reticulum.

In the majority of cells, _vß₃-induced [Ca²⁺]_i increases first occurred at the cell margins and then spread centrally. This centripetal progression was not a phenotypic characteristic because histamine-induced [Ca²⁺]_i increases occurred more uniformly. Although mechanisms are unclear, the peripheral initiation of the response may be in part due to the reported peripheral location of _vß₃.^{31 32} We confirmed _vß₃ aggregation through immunofluorescence (Figure 1). Light fixation of cells and permeabilization improved the detection of immunofluorescence but did not impair the ability of the integrin to undergo aggregation. Lawson et al²³ also reported clustering of the _vß₃ integrin in lightly fixed cells. With the use of confocal microscopy, the immunofluorescent aggregates were best viewed on the luminal surface of the monolayer, indicating that the soluble ligands used probably aggregated luminal integrins. This may be expected because abluminal, matrix-facing integrins were probably already ligated to immobilized matrix elements.³ The aggregates were also located in large part at the cell periphery. Because cell contraction induced by [Ca²⁺]_i-dependent processes opens interendothelial junctions,³³ peripherally located _vß₃ integrins may promote endothelial monolayer permeability through the localization of [Ca²⁺]_i increases to endothelial junctional regions.

[Ca²⁺]_i responses to the _vß₃ integrin have been reported previously in freshly seeded, migrating endothelial cells.⁸ However, these reported responses differ from the present data in at least 2 important respects. First, in migrating cells, [Ca²⁺]_i increases resulted entirely from the entry of external Ca²⁺, whereas here we determined a significant role for intracellular Ca²⁺ release. Second, migrating cells reached a peak response much more gradually, with delays of 20 to 30 minutes after plating, whereas here peak responses occurred in <2 minutes. These differences may be due to the fact that in freshly seeded cells, integrin interactions are probably determined by the extent and speed of cell spreading, which may prolong the [Ca²⁺]_i response. The present short [Ca²⁺]_i transients in stable endothelial cell monolayers may be more representative of vascular _vß₃ responses to circulating ligands.

Our findings address the role of protein tyrosine phosphorylation in receptor-mediated [Ca²⁺]_i regulation. In addition to integrins and growth factor receptors, G protein–linked receptors also activate tyrosine kinases.^{13 14} In the presence of external Ca²⁺ depletion, the addition of MAPTAM or thapsigargin decreased tyrosine phosphorylation on several bands of <46 kDa. A similar endothelial effect was reported for bradykinin, which ligates a G protein–linked receptor.¹³ G protein–linked receptors are likely to first increase [Ca²⁺]_i via the PLC-ß/InsP₃ mechanism, which is tyrosine kinase independent.¹² However, this [Ca²⁺]_i increase may activate Ca²⁺-sensitive tyrosine kinases. The ensuing tyrosine phosphorylation may further increase [Ca²⁺]_i via the PLC-InsP₃ mechanism.¹² In this situation, genistein is expected to attenuate but not completely block the [Ca²⁺]_i response, because it blocks only the tyrosine phosphorylation–dependent [Ca²⁺]_i increase. Accordingly, we confirmed that unlike the complete inhibition of the [Ca²⁺]_i increase to _vß₃ cross-linking, genistein attenuated only the [Ca²⁺]_i increase to histamine.³⁴

Our findings are also relevant to a consideration of the potential role of the endothelial _vß₃ integrin in general vascular responses. The present rapidly developed [Ca²⁺]_i peak and subsequent [Ca²⁺]_i oscillations are characteristic of inflammatory receptors. [Ca²⁺]_i transients elicit processes leading to many types of cell function, such as secretion, endocytosis, and cell contraction.^{11 35 36} In endothelial cells, such transients have been associated with barrier deterioration,³³ leukocyte adhesion,³⁷ and cytokine secretion.⁶ In previous experiments, we reported increases in lung capillary permeability that are attributable to ligation of the _vß₃ integrin.⁴ D’Angelo et al¹⁰ recently reported that the integrin induces vasodilatation. The _vß₃ integrin recognizes apoptotic neutrophils³⁸ and could contribute to endothelial margination of neutrophils.

These considerations indicate that although the biological significance of the endothelial _vß₃ integrin is usually discussed in relation to angiogenesis and vascular growth,² the integrin may play a distinct role in vascular pathophysiological processes in general. Such a role may be particularly important in the lung, which appears to be an exception among vascular beds in that it expresses the _vß₃ integrin under resting, nonproliferative conditions.^{4 39} The _vß₃ integrin binds a wide range of both vitronectin-linked and nonlinked substances of potential pathophysiologic importance, such as SC5b-9, viruses, the thrombin-antithrombin complex, and malignant cells.^{17 21 22 40} This promiscuous binding property across the vast vascular surface of the lung, together with its ability to rapidly mobilize Ca²⁺, accords the endothelial _vß₃ integrin a pathological potential that warrants further study.

	Acknowledgments

 
This work was supported by grants HL-36024 and HL-57556 (Dr J. Bhattacharya) and HL-54157 (Dr S. Bhattacharya) from the National Institutes of Health (Bethesda, Md).

Received July 13, 1999; accepted November 12, 1999.

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