Ca2(+)-dependent, ATP-induced conversion of the [3H]hemicholinium-3 binding sites from high- to low-affinity states in rat striatum: effect of protein kinase inhibitors on this affinity conversion and synaptosomal choline transport.
Tritium-labeled hemicholinium-3 ([3H]HC-3) was used to characterize the sodium-dependent high-affinity choline carrier sites in rat striatal preparations. In an earlier study, we had shown that [3H]HC-3 labels choline carrier sites with high and low affinities and had suggested that the low-affinity sites represent "functional" carrier sites. The objective of the present study was to examine the mechanisms involved in the regulation of the two affinity states of [3H]HC-3 binding. Here, we demonstrate that these two affinity states are totally interconvertible; addition of 0.1 mM ATP in the binding assay medium quantitatively converted all the binding sites to the low-affinity state, whereas addition of 1 mM beta,gamma-methylene 5'-ATP quantitatively converted all the binding sites to the high-affinity state. Preincubation of the tissue (for 15 min at 37 degrees C) before the binding assay also converted the binding sites to the high-affinity state, whereas supplementation of the assay medium with ATP (0.5 mM) again induced expression of the low-affinity state of the binding sites. This effect of ATP was found to be selective for this nucleotide. Neither ADP (1 mM) nor cyclic AMP could mimic such an effect. Other nucleotide triphosphates--CTP (0.5 mM) and GTP (0.5 mM)--also could not substitute for ATP. GTP, however, caused nearly a 35% reduction in the number of binding sites, accompanying a loss of the low-affinity component of binding. This effect of GTP was also shared by 5'-guanylylimidodiphosphate but not by GDP or cyclic GMP. This ATP-dependent low-affinity conversion of [3H]HC-3 binding sites requires divalent metal ions.(ABSTRACT TRUNCATED AT 250 WORDS)
Chatterjee
T K
TK
Department of Pharmacology, College of Medicine, University of Iowa, Iowa City 52242.
Bhatnagar
R K
RK
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology*; D002794:Choline / Q000378:metabolism; D003342:Corpus Striatum / Q000378:metabolism*; D006072:Gossypol / Q000494:pharmacology; D006426:Hemicholinium 3 / Q000378:metabolism*; D008297:Male; D047428:Protein Kinase Inhibitors; D051381:Rats; D011919:Rats, Inbred Strains; D013574:Synaptosomes / Q000378:metabolism; D014316:Tritium
D047428:Protein Kinase Inhibitors; D014316:Tritium; D006426:Hemicholinium 3; D000255:Adenosine Triphosphate; D006072:Gossypol; D002794:Choline; D002118:Calcium