title: nitric oxide triggers the phosphatidylinositol 3-kinase/Akt survival pathway in insulin-producing RINm5F cells by arousing Src to activate insulin receptor substrate-1.
abstract: Mechanisms involved in the protective action of nitric oxide (NO) in insulin-producing cells are a matter of debate. We have previously shown that pharmacological inhibition of c-Src cancels the antiapoptotic action of low and sustained concentrations of exogenous NO. In this study, using insulin-producing RINm5F cells that overexpress Src either permanently active (v-Src) or dominant negative (dn-Src) forms, we determine that this tyrosine kinase is the principal mediator of the protective action of NO. We also show that Src-directed activation of insulin receptor substrate-1, phosphatidylinositol 3-kinase (PI3K), Akt, and Bad phosphorylation conform a substantial component of the survival route because pharmacological inhibition of PI3K and Akt canceled the antiapoptotic effects of NO. Studies performed with the protein kinase G (PKG) inhibitor KT-5823 revealed that NO-dependent activation of c-Src/ insulin receptor substrate-1 is not affected by PKG activation. By contrast, Akt and Bad activation are partially dependent on PKG activation. Endogenous production of NO after overexpression of endothelial nitric oxide synthase in RINm5F cells mimics the effects produced by generation of low amounts of NO from exogenous diethylenetriamine/NO. In addition, we found that NO produces c-Src/PI3K- and PKG-dependent activation of ERK 1/2. The MAPK kinase inhibitor PD 98059 suppresses NO-dependent protection from DNA fragmentation induced by serum deprivation. The protective action of low and sustained concentration of NO is also observed in staurosporine- and Taxol-induced apoptosis. Finally, NO also protects isolated rat islets from DNA fragmentation induced by serum deprivation. These data strengthen the notion that NO production at physiological levels plays a role in protection from apoptosis in pancreatic beta-cells.
authors.affiliation: Laboratory of Biochemistry of the Immune System, Department of Medical Biochemistry and Molecular Biology, University of Sevilla, 41009 Sevilla, Spain.
authors.forename: Juan R
authors.initials: JR
authors.lastname: Tejedo
authors.forename: Gladys M
authors.initials: GM
authors.lastname: Cahuana
authors.forename: Remedios
authors.initials: R
authors.lastname: Ramírez
authors.forename: Margarida
authors.initials: M
authors.lastname: Esbert
authors.forename: Juan
authors.initials: J
authors.lastname: Jiménez
authors.forename: Francisco
authors.initials: F
authors.lastname: Sobrino
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Bedoya
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D002470:Cell Survival; D016895:Culture Media, Serum-Free; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D055504:Insulin Receptor Substrate Proteins; D007515:Islets of Langerhans / Q000378:metabolism*; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009569:Nitric Oxide / Q000494:pharmacology* / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000235:genetics; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010750:Phosphoproteins / Q000502:physiology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017208:Rats, Wistar; D019061:src-Family Kinases
chemical_list: D000081247:CSK Tyrosine-Protein Kinase; D016895:Culture Media, Serum-Free; D017869:Cyclic GMP-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D055504:Insulin Receptor Substrate Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019061:src-Family Kinases; C526296:Irs1 protein, rat; C496316:Nos3 protein, rat; C494920:Akt1 protein, rat