alpha(v)beta(3) integrin induces tyrosine phosphorylation-dependent Ca(2+) influx in pulmonary endothelial cells.
The endothelial alpha(v)beta(3) 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 Ca(2+) concentration ([Ca(2+)](i)) according to the Fura 2 ratio imaging method in single cells of confluent monolayers. At baseline, endothelial [Ca(2+)](i) levels remained steady at 86 nmol/L for >20 minutes. Cross-linking of the alpha(v)beta(3) integrin through the sequential exposure of monolayers to anti-alpha(v)beta(3) monoclonal antibody LM609 and secondary IgG resulted in a [Ca(2+)](i) increase of 100% above baseline. This increase commenced in <0.5 minute, peaked in <2 minutes, and decayed to baseline in approximately 5 minutes. Similar responses occurred after the addition of vitronectin (400 microg/mL). In contrast, external Ca(2+) depletion blunted the cross-linking-induced [Ca(2+)](i) increase by 60%, a response that was completely inhibited when the monolayers were also pretreated with thapsigargin. Thus, the [Ca(2+)](i) increase was attributable in part to the release of Ca(2+) from endosomal stores but mostly to Ca(2+) influx across the plasma membrane. Induced aggregation of the alpha(v)beta(3) integrin enhanced tyrosine phosphorylation of phospholipase C-gamma1 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 alpha(v)beta(3)-induced [Ca(2+)](i) increases. We conclude that aggregation of the endothelial alpha(v)beta(3) integrin induces a rapid tyrosine phosphorylation-dependent increase in [Ca(2+)](i). This response may subserve the inflammatory role of alpha(v)beta(3) integrin in blood vessels.
Bhattacharya
S
S
Departments of Pediatrics and Medicine, St Luke's-Roosevelt Hospital Center, New York, NY 10019, USA.
Ying
X
X
Fu
C
C
Patel
R
R
Kuebler
W
W
Greenberg
S
S
Bhattacharya
J
J
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D003600:Cytosol / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D009994:Osmolar Concentration; D010766:Phosphorylation; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism*; D019030:Receptors, Vitronectin / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism*
D019030:Receptors, Vitronectin; D014443:Tyrosine; D002118:Calcium