Inhibition of Glial Cell Line‐Derived Neurotrophic Factor Induced Intracellular Activity by K‐252b on Dopaminergic Neurons

: 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 µM K‐252b decreased the efficacy of GDNF by 60%. Concentrations of GDNF 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.