title: Inhibition of glial cell line-derived neurotrophic factor induced intracellular activity by K-252b on dopaminergic neurons.
abstract: The c-ret protooncogene encodes Ret, the functional tyrosine kinase receptor for glial cell line-derived neurotrophic factor (GDNF). K-252b, a known protein tyrosine kinase inhibitor, has been shown earlier to inhibit the trophic activity of brain-derived neurotrophic factor on dopaminergic (DAergic) neurons and nerve growth factor on basal forebrain cholinergic neurons while potentiating neurotrophin-3 activity on central cholinergic and peripheral sensory neurons and PC12 cells. We tested whether K-252b would modulate GDNF-induced differentiation in DAergic neuron cultures. Exposure to 1 ng/ml GDNF increased dopamine (DA) uptake 80% above control, whereas treatment with 5 microM K-252b decreased the efficacy of GDNF by 60%. Concentrations of GDNF of <100 pg/ml were completely inhibited, whereas concentrations of >100 pg/ml were moderately active, between 10 and 20% above control. In addition, K-252b shifted the ED50 from 20 to 200 pg/ml. GDNF treatment increased soma size and neurite outgrowth in tyrosine hydroxylase-immunoreactive neurons. K-252b inhibited differentiation of these morphological parameters induced by GDNF. Furthermore, GDNF stimulated Ret autophosphorylation at maximal levels, whereas the inhibition of DA uptake and morphological differentiation by K-252b correlated with a significantly decreased level of Ret autophosphorylation. Therefore, K-252b is able to inhibit intracellular activities induced by GDNF on mesencephalic DAergic neurons.
authors.affiliation: Department of Neuroscience, Amgen, Inc., Thousand Oaks, California 91320, U.S.A.
authors.forename: K
authors.initials: K
authors.lastname: Pong
authors.forename: R Y
authors.initials: RY
authors.lastname: Xu
authors.forename: K D
authors.initials: KD
authors.lastname: Beck
authors.forename: T J
authors.initials: TJ
authors.lastname: Zhang
authors.forename: J C
authors.initials: JC
authors.lastname: Louis
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002227:Carbazoles / Q000494:pharmacology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D004298:Dopamine / Q000378:metabolism*; D029721:Drosophila Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005333:Fetus; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D006801:Humans; D026121:Indole Alkaloids; D007700:Kinetics; D008636:Mesencephalon / Q000378:metabolism*; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018696:Neuroprotective Agents / Q000494:pharmacology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051096:Proto-Oncogene Proteins c-ret; D051381:Rats; D017207:Rats, Sprague-Dawley; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000494:pharmacology; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis
chemical_list: D002227:Carbazoles; D029721:Drosophila Proteins; D004791:Enzyme Inhibitors; C495039:GDNF protein, human; C495041:Gdnf protein, rat; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D026121:Indole Alkaloids; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D018696:Neuroprotective Agents; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; C049985:staurosporine aglycone; D014446:Tyrosine 3-Monooxygenase; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C416649:Ret protein, Drosophila; C495001:Ret protein, rat; D011493:Protein Kinase C; D004298:Dopamine