Effects of phorbol esters and secretagogues on nitrobenzylthioinosine binding to nucleoside transporters and nucleoside uptake in cultured chromaffin cells.
Secretagogues inhibited adenosine uptake in chromaffin cells without causing apparent changes in the uptake affinity. The inhibition caused by carbachol, nicotine and acetylcholine reached 50%. This inhibition was reproduced by the action of protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA; 100 nM), phorbol 12,13-dibutyrate (PDBu; 100 nM), dicaproin (10 micrograms/ml) and tricaprylin (10 micrograms/ml), with inhibitions of Vmax. of 18, 20, 37 and 47% respectively. No changes in the affinity of uptake were observed with these effectors. Down-regulation of protein kinase C by phorbol esters decreased the inhibitory effects of carbachol on adenosine uptake. Binding studies with nitrobenzylthioinosine (NBTI) showed a similar decrease in the number of transporters when chromaffin cells were treated with the same effectors used for the uptake studies. The high-affinity dissociation constants showed minor changes with respect to the control. The ratio between maximal uptake capacity and the transporter number per cell was not significantly modified by the action of secretagogues or direct effectors of protein kinase C. The number of high-affinity binding sites for NBTI was decreased in cellular homogenates by the direct action of protein kinase C activators, with staurosporine able to reverse this action. Protein kinase C from bovine brain in the presence of ATP and effectors, decreased the number of high-affinity NBTI-binding sites in purified chromaffin cell plasma membranes. These data suggest the possibility of a molecular modification at the transporter level.
Delicado
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EG
Departamento de Bioquímica, Facultad de Veterinaria Universidad Complutense de Madrid, Spain.
Sen
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RP
Miras-Portugal
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MT
D000241:Adenosine / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002838:Chromaffin System / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism*; D033703:Nucleoside Transport Proteins; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000737:chemistry
D002352:Carrier Proteins; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D013868:Thioinosine; D011493:Protein Kinase C; C001789:4-nitrobenzylthioinosine; D000241:Adenosine; D013755:Tetradecanoylphorbol Acetate