Flow cytometric studies of nucleoside transport regulation in single chromaffin cells.
The present paper reveals that a fluorescent derivative of nitrobenzylthioinosine, 5-(SAENTA-x8)-fluorescein, is a highly specific inhibitor of the neural NBTI-sensitive nucleoside transporter. 5-(SAENTA-x8)-fluorescein inhibited adenosine transport and [3H]NBTI binding with a Ki of 4 nM in cultured chromaffin cells. Flow cytometry demonstrated that 5-(SAENTA-x8)-fluorescein specifically interacted with the NBTI-sensitive nucleoside transporters with high affinity (K[D] = 6 nM). Activation 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 transport in single cells from heterogeneous neural cell populations.
Sen
R P
RP
Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain.
Delicado
E G
EG
Alvarez
A
A
Brocklebank
A M
AM
Wiley
J S
JS
Miras-Portugal
M T
MT
D000241:Adenosine / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000378:metabolism*; D005434:Flow Cytometry; D005452:Fluoresceins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D033703:Nucleoside Transport Proteins; D011684:Purine Nucleosides / Q000378:metabolism*; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism
D002352:Carrier Proteins; D005452:Fluoresceins; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D011684:Purine Nucleosides; C079645:5-(SAENTA-x8)fluorescein; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine; D000241:Adenosine