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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—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

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