title: Binding of [3H]dihydrotetrabenazine and [125I]azidoiodoketanserin photoaffinity labeling of the monoamine transporter of platelet 5-HT organelles.
abstract: The carrier for 5-hydroxytryptamine (5-HT) of the 5-HT storage organelles of blood platelets was characterized by [3H]dihydrotetrabenazine binding and [125I]azidoiodokentanserin photoaffinity labeling. [3H]Dihydrotetrabenazine bound with high affinity to membrane preparations from different animal species. The [3H]dihydrotetrabenazine Bmax value was about 10-fold higher in rabbit (9.4 +/- 1.3 pmol/mg protein) than in human, rat and guinea-pig preparations (Bmax values = 1.1 +/- 0.2, 1.2 +/- 0.1 and 0.52 +/- 0.06 pmol/mg protein, respectively). After rabbit platelet subcellular fractionation, [3H]dihydrotetrabenazine binding was highly enriched in the fraction corresponding to pure 5-HT organelles, whereas ligand binding was much lower in the other subcellular fractions. Conversely, [3H]paroxetine binding sites were more concentrated in the lower density fractions, with no binding to the 5-HT granules. In competition experiments, [3H]dihydrotetrabenazine binding to human platelet membranes and rabbit platelet 5-HT organelles was markedly inhibited by the benzo[a]quinolizine derivatives, tetrabenazine and Ro 4-1284, and by ketanserin. In isolated rabbit platelet 5-HT organelles, reserpine showed a relatively high IC50 (930 nM), but the presence of ATP increased its potency about 10-fold. Paroxetine, methysergide and carrier substrates had little or no effect. After photoaffinity labeling of rabbit 5-HT granules with [125I]azidoiodoketanserin, the radioactivity was incorporated into several polypeptides. The presence of Ro 4-1284, reserpine and ketanserin prevented the labeling of a polypeptide of 85 kDa. The data obtained suggest that this protein represents a component of the granular carrier which binds [3H]dihydrotetrabenazine.
authors: Department of Pharmacology, University of Milan, Italy.
authors: A M
authors: AM
authors: Cesura
authors: B
authors: B
authors: Bertocci
authors: M
authors: M
authors: Da Prada
mesh_terms: D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001386:Azides; D015306:Biogenic Monoamines / Q000097:blood*; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006168:Guinea Pigs; D006801:Humans; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D007650:Ketanserin / Q000031:analogs & derivatives*; D017374:Paroxetine; D010777:Photochemistry; D010880:Piperidines / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011985:Receptors, Serotonin / Q000187:drug effects / Q000378:metabolism*; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D013747:Tetrabenazine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D001386:Azides; D015306:Biogenic Monoamines; D001798:Blood Proteins; D007457:Iodine Radioisotopes; D010880:Piperidines; D011985:Receptors, Serotonin; C067115:azidoiodoketanserin; C032811:dihydrotetrabenazine; D017374:Paroxetine; D007650:Ketanserin; D013747:Tetrabenazine