Identification of dopamine plasma membrane and vesicular transporters in human peripheral blood lymphocytes

Plasma membrane dopamine transporter (DAT), vesicular monoamine transporter s (VMAT) type-1 and -2 and the expression of the dopaminergic markers dopam ine and tyrosine hydroxylase were assessed in membranes and/or in cytospin centrifuged human peripheral blood lymphocytes. The radiolabeled DAT ligand [H-3]GBR12935 was bound to peripheral lymphocytes in a manner consistent w ith the specific binding to a dopamine uptake system, with a dissociation c onstant similar to that found in striatum, but with a lower density of bind ing sites. On the other hand, no specific binding occurred in cerebellum us ed as a test tissue not expressing DAT. Western blot analysis using antibod ies raised against amino or carboxy terminus of DAT or against VMAT-1 or VM AT-2 revealed labeling of single bands of approximately 76, 55 or 68 KDa, r espectively, displaying similar migration characteristics in lymphocytes an d test tissues used for comparison. Immunofluorescence revealed that anti-d opamine, anti-tyrosine hydroxylase, anti-DAT, anti-VMAT-1 and anti-VMAT-2 a ntibodies labeled the total population of cytospin-centrifuged lymphocytes mounted on microscope slides. Confocal laser microscopy demonstrated that d opamine and VMAT-2 immunoreactivity was developed mainly in cytoplasmic pun ctiform areas likely corresponding to vesicles and to a lower extent was as sociated to plasma membrane. Tyrosine hydroxylase immunoreactivity was diff used to cytoplasm and to plasma membrane of lymphocytes, whereas DAT and VM AT-1 immunoreactivity were located almost exclusively in lymphocyte plasma membrane and cytoplasm, respectively. Lymphocyte DAT characterized in this study has probably functional relevance as [H-3]dopamine was taken up by in tact lymphocytes and uptake was inhibited specifically by compounds known t o affect dopamine transport. These findings indicate that human peripheral blood lymphocytes possess DAT plasma membrane and VMAT-1 and VMAT-2 transpo rters. Increasing evidence indicates that dopamine transporter changes may be related to neuronal injury. In view of this assessment of lymphocyte DAT and VMAT transporters can be considered for identifying pathologies charac terized by impaired dopaminergic neurotransmission. (C) 2001 Elsevier Scien ce B.V. All rights reserved.