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 re ceptor 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 d opaminergic (DAergic) neurons and nerve growth factor on basal forebra in cholinergic neurons while potentiating neurotrophin-3 activity on c entral cholinergic and peripheral sensory neurons and PC12 cells. We t ested whether K-252b would modulate GDNF-induced differentiation in DA ergic neuron cultures. Exposure to 1 ng/ml GDNF increased dopamine (DA ) uptake 80% above control, whereas treatment with 5 mu M K-252b decre ased 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-25 2b shifted the ED50 from 20 to 200 pg/ml. GDNF treatment increased som a size and neurite outgrowth in tyrosine hydroxylase-immunoreactive ne urons. K-252b inhibited differentiation of these morphological paramet ers induced by GDNF. Furthermore, GDNF stimulated Ret autophosphorylat ion at maximal levels, whereas the inhibition of DA uptake and morphol ogical differentiation by K-252b correlated with a significantly decre ased level of Ret autophosphorylation. Therefore, K-252b is able to in hibit intracellular activities induced by GDNF on mesencephalic DAergi c neurons.