Multiple Roles of Protein Kinase A in Arachidonic Acid-Mediated Ca2+ Entry and Tumor-Derived Human Endothelial Cell Migration

We recently showed that arachidonic acid (AA) triggers calcium signals in endothelial cells derived from human breast carcinoma (B-TEC). In particular, AA-dependent Ca2+ entry is involved in the early steps of tumor angiogenesis in vitro. Here, we investigated the multiple roles of the nitric oxide (NO) and cyclic AMP/protein kinase A (PKA) pathways in AA-mediated Ca2+ signaling in the same cells. B-TEC stimulation with 5 mu mol/L AA resulted in endothelial NO synthase (NOS) phosphorylation at Ser(1177), and NO release was measured with the fluorescent NO-sensitive probe DAR4M-AM. PKA inhibition by the use of the membrane-permeable PKA inhibitory peptide myristoylated PKI14-22 completely prevented both AA-and NO-induced calcium entry and abolished B-TEC migration promoted by AA. AA-dependent calcium entry and cell migration were significantly affected by both the NOS inhibitor N-G-nitro-L-arginine methyl ester and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, suggesting that NO release is functionally involved in the signaling dependent on AA. Moreover, pretreatment with carboxyamidotriazole, an antiangiogenic compound that interferes with agonist-activated calcium entry, prevented AA-dependent B-TEC motility. Interestingly, even in the absence of AA, enhancement of the cyclic AMP/PKA pathway with the adenylyl cyclase activator forskolin evoked a calcium entry dependent on NOS recruitment and NO release. The functional relevance of AA-induced calcium entry could be restricted to tumor-derived endothelial cells (EC) because AA evoked a smaller calcium entry in normal human microvascular ECs compared with B-TECs, and even more importantly, it was unable to promote cell motility in wound healing assay. This evidence opens an intriguing opportunity for differential pharmacologic treatment between normal and tumor-derived human ECs. Mol Cancer Res; 8(11); 1466-76. (C) 2010 AACR.