Immediate and delayed VEGF-mediated NO synthesis in endothelial cells: role of PI3K, PKC and PLC pathways.
1. The mechanism(s) by which vascular endothelial growth factor (VEGF) induces endothelial nitric oxide synthase (eNOS) activation remain(s) unclear up to a certain extent. Therefore, we sought to evaluate the contribution of numerous pathways in VEGF-induced nitric oxide (NO) synthesis by measuring cGMP production. In addition, as VEGF induces the synthesis of NO and platelet-activating factor (PAF), we wanted to assess if the induction of PAF and NO is contributing to the synthesis of each other. 2. Herein, we show that a treatment of endothelial cells with a phospholipase C (PLC) inhibitor (U73122), a calmodulin antagonist (W-7) or with intracellular calcium chelators (EGTA/AM, BAPTA/AM) prevented VEGF-mediated eNOS Ser(1177)-phosphorylation and NO synthesis measured by cGMP production. 3. Pretreatment with phosphatidylinositol 3-kinase (PI3K) (Wortmannin, LY294002) or protein kinase C (PKC) (GF109203X, Ro318220) inhibitors attenuated eNOS Ser(1177)-phosphorylation mediated by VEGF, but did not alter immediate (0-10 min) cGMP synthesis induced by VEGF, but abrogated by up to 84% the delayed (10-30 min) cGMP synthesis. 4. Pretreatment with PAF synthesis inhibitors or with PAF receptor antagonists did not abrogate neither eNOS Ser(1177)-phosphorylation nor cGMP synthesis mediated by VEGF. 5. In conclusion, VEGF induces an immediate cGMP synthesis through the PLC-Ca2+/CaM pathway, and that the induction of delayed cGMP synthesis implies Akt and PKC activity.
Gélinas
David S
DS
Department of Pharmacology, University of Montreal, Montreal (Qc), Canada.
Bernatchez
Pascal N
PN
Rollin
Simon
S
Bazan
Nicolas G
NG
Sirois
Martin G
MG
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000037:antagonists & inhibitors; D002417:Cattle; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D002867:Chromones / Q000494:pharmacology; D006152:Cyclic GMP / Q000378:metabolism; D004224:Diterpenes; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D016228:Endothelial Growth Factors / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D046934:Ginkgolides; D007211:Indoles / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D007476:Ionophores / Q000494:pharmacology; D007783:Lactones / Q000494:pharmacology; D008222:Lymphokines / Q000494:pharmacology*; D008301:Maleimides / Q000494:pharmacology; D009025:Morpholines / Q000494:pharmacology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D009599:Nitroprusside / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D010972:Platelet Activating Factor / Q000378:metabolism / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000037:antagonists & inhibitors; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors; D043562:Receptors, G-Protein-Coupled; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013997:Time Factors; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D000077191:Wortmannin
D015056:1-Methyl-3-isobutylxanthine; D000730:Androstadienes; D000001:Calcimycin; D002118:Calcium; D002614:Chelating Agents; D002867:Chromones; D006152:Cyclic GMP; D004224:Diterpenes; D004533:Egtazic Acid; D016228:Endothelial Growth Factors; D004791:Enzyme Inhibitors; D046934:Ginkgolides; D007211:Indoles; D036341:Intercellular Signaling Peptides and Proteins; D007476:Ionophores; D007783:Lactones; D008222:Lymphokines; D008301:Maleimides; D009025:Morpholines; D019331:NG-Nitroarginine Methyl Ester; D009569:Nitric Oxide; D020030:Nitric Oxide Donors; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D009599:Nitroprusside; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D012694:Serine; D010738:Type C Phospholipases; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D000077191:Wortmannin; C039351:platelet activating factor receptor; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C045856:ginkgolide B; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; C070515:bisindolylmaleimide I; C064758:Ro 31-8220