During angiogenesis, endothelial cells react to stimulation with finely tuned signaling responses. The role of calcium-regulated signaling in angiogenesis has not been defined. This study investigated the calcium dependency of endothelial cell proliferation and invasion by using an inhibitor of ligand-stimulated calcium influx, CAI (carboxyamidotriazole). Incubation with CAI significantly inhibited proliferation of human umbilical vein endothelial cells (HUVECs) in response to serum (IC50 = 1 ,uM) or basic fibroblast growth factor (FGF2; P2 < 0.005 at 10 ,uM). Statistically significant inhibition of HUVEC adhesion and motility to basement membrane proteins laminin, fibronectin, and type IV collagen was demonstrated (adhesion, P2 < 0.004-0.01; motility, P2 < 0.009-0.018). Marked inhibition of native and FGF2-induced gelatinase activity was shown by zymogram analysis and was confirmed by Northern blot analysis. CAI inhibited HUVEC tube formation on Matrigel and inhibited in vivo angiogenesis in the chicken chorioallantoic membrane assay, 67% at 20 ,LM and 56% at 10 ,uM compared with 16% for an inactive CAI analog or 9%o for 0.1% dimethyl sulfoxide control. Incubation of HUVECs with CAI and/or FGF2 followed by immunoprecipitation with anti-phosphotyrosine antibody showed inhibition of FGF2-induced tyrosine phosphorylation of proteins in the range 110-150 kDa. These results suggest that calcium-regulated events are important in native and FGF2-stimulated HUVEC proliferation and invasion, perhaps through regulation of FGF2-induced phosphorylation events, and indicate a role for calcium in the regulation of angiogenesis in vivo.

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Activation of the muscarinic receptor in Chinese hamster ovary (CHO) cells results in a reversal of the malignant phenotype for which spreading into a bipolar, fibroblastic morphology is a marker. The process of morphologic change requires multiple events, including alterations in adhesions to substrates and cytoskeletal re-arrangement. In this report, we demonstrate the calcium-dependent involvement of p125 FAK in this cellular shape change using an inhibitor of ligand-induced calcium influx, carboxyamido-triazole (CAI). p125 FAK becomes tyrosine-phosphorylated after exposure to the agonist carbachol (CC), reaching maximal phosphorylation prior to initiation of cellular shape change at 1 hr into CC exposure (386 6 103%). Phosphorylation remained elevated through the shape change (4-12 hr The Chinese hamster ovary (CHO) m5 muscarinic acetylcholine receptor (m5AChR) tumor-suppressor model is a unique model in which to study spreading-related signal-transduction events . CHO cells stably transfected with m5AChR undergo a morphologic change from stellate to fibroblastic when stimulated with the specific muscarinic agonist carbachol (CC). This shape change is associated with a marked reduction in the ability of CHOm5 cells to form tumors in nude mice . The m5AChR is coupled to multiple signal-transduction pathways, including membrane-associated phospholipases (C-b, C-g, A2 and D), release of internal Ca 21 stores, stimulation of Ca 21 influx through receptor-operated Ca 21 channels and activation of adenylyl cyclase (Felder, 1995;Felder et al., 1993;Gusovsky et al., 1993). The shape change was shown to be dependent upon m5AChR-mediated mobilization of intracellular Ca 21 or cAMP (Felder et al., , 1994Singer-Lahat et al., 1996). We have shown that this calcium influx can be inhibited by concomitant exposure to carboxyamido-triazole (CAI), an inhibitor of nonvoltage-gated calcium influx, at CAI concentrations that inhibit migration, proliferation and angiogenesis (Felder et al., 1991Kohn et al., 1994aKohn et al., , 1995. CAI blockade of CC-mediated calcium influx prevented the phenotypic change of CHOm5 cells . This spreading during the shape change provides a unique model in which to investigate the temporal nature and Ca 21 sensitivity of the signaling events underlying this tumor-suppression model.Cells interact with their extracellular environment, the extracellular matrix and basement membrane, through cell-surface contacts termed focal adhesions. Focal-adhesion contacts contain regions of enhanced tyrosine phosphorylation through which cytoskeletal elements may couple to membrane-associated signal-transducing pathways (Hansen et al., 1994;Maher et al., 1985). These points of contact are regulated in a complex fashion involving cell interaction with the extracellular substratum and subsequent outside-in signaling to direct cellular re-organization. Down-stream effector events are also complex, involving several signaling cascades, such as mobilization of intracellular Ca 21 , phosphorylation and generation of leu...