FLOW CYTOMETRIC STUDIES OF NUCLEOSIDE TRANSPORT REGULATION IN SINGLE CHROMAFFIN CELLS

The present paper reveals that a fluorescent derivative of nitrobenzyl thioinosine, 5-(SAENTA-x8)-fluorescein, is a highly specific inhibitor of the neural NBTI-sensitive nucleoside transporter, 5-(SAENTA-x8)-fl uorescein inhibited adenosine transport and [H-3]NBTI binding with a K -i of 4 nM in cultured chromaffin cells. Flow cytometry demonstrated t hat 5-(SAENTA-x8)-fluorescein specifically interacted with the NBTI-se nsitive nucleoside transporters with high affinity (K-D = 6 nM). Activ ation of protein kinases A and C with forskolin or nicotinic receptor agonists, respectively, resulted in 50% inhibition of the fluorescence bound to the cells. Flow cytometry will allow studying nucleoside tra nsport in single cells from heterogeneous neural cell populations. (C) 1998 Federation of European Biochemical Societies.