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1.
J Pharm Sci ; 101(1): 154-63, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21905028

RESUMO

The sodium-coupled oligopeptide transporters 1 and 2 (SOPT1 and SOPT2) transport peptides consisting of at least five amino acids and show potential for the delivery of therapeutically relevant peptides/peptidomimetics. Here, we examined the expression of these two transporters in the retinal neuronal cell line RGC-5. These cells showed robust uptake activity for the synthetic pentapeptide DADLE ([D-Ala(2),D-Leu(5)]-Enkephalin). The uptake was Na(+) dependent and saturable (K(t), 6.2 ± 0.6 µM). A variety of oligopeptides inhibited DADLE uptake. The uptake of the competing oligopeptides was directly demonstrated with fluorescein isothiocyanate-labeled Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys in RGC-5 cells and primary mouse retinal ganglion cells. The characteristics of DADLE uptake matched those of SOPT2. We then examined the expression of SOPT1 in these cells with deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH(2)) as the substrate and found that RGC-5 cells also expressed SOPT1. As it is already known that SOPT1 is expressed in the neuronal cell line SK-N-SH, we investigated SOPT2 expression in these cells to determine whether the presence of both oligopeptide transporters is a common feature of neuronal cells. These studies showed that SK-N-SH cells also expressed SOPT2. This constitutes the first report on the functional characterization of SOPT1 and SOPT2 in retinal neuronal cells and on the expression of SOPT2 in nonretinal neuronal cells.


Assuntos
Leucina Encefalina-2-Alanina/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Peptídeos Opioides/metabolismo , Células Ganglionares da Retina/metabolismo , Aminoácidos/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Cinética , Camundongos , Oligopeptídeos/metabolismo , Sódio/metabolismo , Especificidade por Substrato
2.
Biochim Biophys Acta ; 1798(6): 1164-71, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20211600

RESUMO

Pyroglutamate, also known as 5-oxoproline, is a structural analog of proline. This amino acid derivative is a byproduct of glutathione metabolism, and is reabsorbed efficiently in kidney by Na(+)-coupled transport mechanisms. Previous studies have focused on potential participation of amino acid transport systems in renal reabsorption of this compound. Here we show that it is not the amino acid transport systems but instead the Na(+)-coupled monocarboxylate transporter SLC5A8 that plays a predominant role in this reabsorptive process. Expression of cloned human and mouse SLC5A8 in mammalian cells induces Na(+)-dependent transport of pyroglutamate that is inhibitable by various SLC5A8 substrates. SLC5A8-mediated transport of pyroglutamate is saturable with a Michaelis constant of 0.36+/-0.04mM. Na(+)-activation of the transport process exhibits sigmoidal kinetics with a Hill coefficient of 1.8+/-0.4, indicating involvement of more than one Na(+) in the activation process. Expression of SLC5A8 in Xenopuslaevis oocytes induces Na(+)-dependent inward currents in the presence of pyroglutamate under voltage-clamp conditions. The concentration of pyroglutamate necessary for induction of half-maximal current is 0.19+/-0.01mM. The Na(+)-activation kinetics is sigmoidal with a Hill coefficient of 2.3+/-0.2. Ibuprofen, a blocker of SLC5A8, suppressed pyroglutamate-induced currents in SLC5A8-expressing oocytes; the concentration of the blocker necessary for causing half-maximal inhibition is 14+/-1microM. The involvement of SLC5A8 can be demonstrated in rabbit renal brush border membrane vesicles by showing that the Na(+)-dependent uptake of pyroglutamate in these vesicles is inhibitable by known substrates of SLC5A8. The Na(+) gradient-driven pyroglutamate uptake was stimulated by an inside-negative K(+) diffusion potential induced by valinomycin, showing that the uptake process is electrogenic.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Ácido Pirrolidonocarboxílico/metabolismo , Sódio/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Membrana Celular/genética , Expressão Gênica , Humanos , Ionóforos/farmacologia , Rim/metabolismo , Cinética , Camundongos , Microvilosidades/genética , Microvilosidades/metabolismo , Transportadores de Ácidos Monocarboxílicos , Oócitos , Técnicas de Patch-Clamp , Potássio/metabolismo , Coelhos , Ratos , Valinomicina/farmacologia , Xenopus laevis
3.
Invest Ophthalmol Vis Sci ; 51(1): 413-20, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19643969

RESUMO

PURPOSE: A sodium-coupled oligopeptide transporter (SOPT1) was described originally in ARPE-19 cells. The transporter is inducible by HIV-1 Tat. Recent studies of conjunctival epithelial cells have identified a second oligopeptide transporter (SOPT2). This study was conducted to determine whether the newly discovered SOPT2 is expressed in ARPE-19 cells, to examine whether the new transporter is also inducible by HIV-1 Tat, and to find out whether this transporter is expressed in primary RPE cells. METHODS: The transport activity of SOPT2 was monitored in control and Tat-expressing ARPE-19 cells and in primary mouse and human fetal RPE cells by the uptake of the synthetic opioid peptide DADLE ((H-Tyr-D-Ala-Gly-Phe-D-Leu-OH) and by its susceptibility to inhibition by small peptides. Substrate selectivity was examined by competition studies and kinetic parameters were determined by saturation analysis. RESULTS: ARPE-19 cells express DADLE uptake activity that is inhibited by small peptides, indicating expression of SOPT2 in these cells. The activity of SOPT2 is induced by HIV-1 Tat. SOPT2 accepts endogenous and synthetic opioid peptides as substrates, but nonpeptide opiate antagonists are excluded. An 11-amino-acid HIV-1 Tat peptide also serves as a high-affinity substrate for the transporter. Primary cultures of mouse and human fetal RPE cells express SOPT2. The transporter is partially Na(+)-dependent with comparable substrate selectivity and inhibitor specificity in the presence and absence of Na(+). CONCLUSIONS: ARPE-19 cells as well as primary mouse and human fetal RPE cells express the newly discovered oligopeptide transporter SOPT2, and the transporter is induced by HIV-1 Tat in ARPE-19 cells.


Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Oligopeptídeos/metabolismo , Peptídeos Opioides/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Sódio/farmacologia , Animais , Transporte Biológico Ativo , Células Cultivadas , Leucina Encefalina-2-Alanina/metabolismo , Humanos , Camundongos , Fragmentos de Peptídeos/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Transfecção , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
4.
Drug Metab Pharmacokinet ; 23(4): 254-62, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18762712

RESUMO

We have recently identified a Na+/Cl--coupled transport system in mammalian cells for endogenous and synthetic opioid peptides. This transport system does not transport dipeptides/tripeptides, but is stimulated by these small peptides. Here we investigated the influence of L-kyotorphin (L-Tyr-L-Arg), an endogenous dipeptide with opioid activity, on this transport system. The activity of the transport system, measured in SK-N-SH cells (a human neuronal cell line) with deltorphin II as a model substrate, was stimulated approximately 2.5-fold by L-kyotorphin, with half-maximal stimulation occurring at approximately 100 microM. The stimulation was associated primarily with an increase in the affinity for deltorphin II. The stimulation caused by L-kyotorphin was stereospecific; L-Tyr-D-Arg (D-kyotorphin) had minimal effect. The influence of L-kyotorphin was observed also in a different cell line which expressed the opioid peptide transport system. While L-kyotorphin is a stimulator of opioid peptide transport, it is a transportable substrate for the H+-coupled peptide transporter PEPT2, which is expressed widely in the brain. Since the activity of the opioid peptide transport system is modulated by extracellular L-kyotorphin and since PEPT2 is an important determinant of extracellular L-kyotorphin in the brain, the expression/activity of PEPT2 may be a critical factor in the modulation of opioidergic neurotransmission in vivo.


Assuntos
Cloretos/metabolismo , Endorfinas/farmacologia , Peptídeos Opioides/metabolismo , Sódio/metabolismo , Simportadores/fisiologia , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Endorfinas/farmacocinética , Humanos , Oligopeptídeos/farmacocinética , Oligopeptídeos/farmacologia , Estereoisomerismo , Xenopus laevis
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