RESUMO
The organic cation transporter (OCT, SLC22) family is a family of polyspecific transmembrane proteins that are responsible for the uptake or excretion of many cationic drugs, toxins, and endogenous metabolites in a variety of tissues. Many of the OCTs have been previously characterized, but there are a number of orphan genes whose functions remain unknown. In this study, two novel rat SLC22 genes, SLC22A17 (BOCT1) and SLC22A23 (BOCT2), were cloned and characterized. Northern blot analysis showed that BOCT1 and BOCT2 mRNA was expressed in a wide variety of tissues. BOCT1 was strongly expressed in brain, primary neurons and brain endothelial cells, with highest expression in choroid plexus. BOCT2 was also abundantly expressed in brain, as well as in liver. To characterize the products of these genes, BOCT1 cDNA was isolated from a rat blood-brain barrier cDNA library, and BOCT2 cDNA was isolated from rat brain capillary and from cultured neurons using PCR techniques. Plasmids expressing BOCT1 and BOCT2 were transfected into HEK-293 cells, as were control cDNAs for OCT1 and OCTN2. Recombinant cell surface protein was verified by western blot and fluorescence microscopy. Transport activity of BOCT1 and BOCT2 was evaluated using radioisotope uptake assays. The OCT1- and OCTN2-expressing cells transported the canonical substrates, 1-methyl-4-phenyl-pyridinium (MPP(+)) and carnitine, respectively. However, BOCT1 and BOCT2-expressing cells did not show transport activity for these substrates or a number of other SLC22 substrates. These novel family members have a nonconserved amino terminus, relative to other OCTs, that may preclude typical SLC22 transport function.
Assuntos
Proteínas de Transporte de Cátions Orgânicos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Primers do DNA , DNA Complementar , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas de Transporte de Cátions Orgânicos/química , RNA Mensageiro/genética , Ratos , Homologia de Sequência de AminoácidosRESUMO
Basal forebrain cholinergic neurons (BFCNs) degenerate in aging and Alzheimer's disease. It has been proposed that estrogen can affect the survival and function of BFCNs. This study characterized primary rat BFCN cultures and investigated the effect of estrogen on high-affinity choline uptake (HACU). BFCNs were identified by immunoreactivity to the vesicular acetylcholine transporter (VAChT) and represented up to 5% of total cells. HACU was measured in living BFCN cultures and differentiated from low-affinity choline uptake by hemicholinium-3 (HC-3) inhibition. A HC-3 concentration curve showed that 0.3 muM HC-3, but not higher concentrations that inhibit LACU, could distinguish the two transport activities. 17-beta-Estradiol treatment increased HACU in some culture preparations that contained non-neuronal cells. Elimination of dividing cells using antimitotic treatments resulted in a lack of estrogen effects on HACU. These results suggest that estrogen may have indirect effects on BFCNs that are mediated through non-neuronal cells.
