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 Ca(2+) 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 Ca(2+) signaling in the same cells. B-TEC stimulation with 5 μmol/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 PKI(14-22) 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.
Fiorio Pla
Alessandra
A
Department of Animal and Human Biology, University of Turin, Via Accademia Albertina 13, 10123 Turin, Italy.
Genova
Tullio
T
Pupo
Emanuela
E
Tomatis
Cristiana
C
Genazzani
Armando
A
Zaninetti
Roberta
R
Munaron
Luca
L
D016718:Arachidonic Acid / Q000378:metabolism / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000098:blood supply* / Q000378:metabolism / Q000473:pathology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects*; D018275:Carcinoma, Lobular / Q000098:blood supply / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects* / Q000502:physiology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects
D005576:Colforsin; D016718:Arachidonic Acid; D009569:Nitric Oxide; D000242:Cyclic AMP; D052250:Nitric Oxide Synthase Type III; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium