Expression of HIV-1 Tat in the retina decreases glutathione levels and increases gamma-glutamyl transpeptidase activity. Tat also upregulates the expression of system xc-. Glutathione levels may be decreased and the expression of xc- enhanced in the retina of patients with HIV-1 infection, leading to oxidative stress and excitotoxicity.
The endogenous opioid peptides enkephalins, dynorphins and endorphins consist of five or more amino acids. These peptides participate in a multitude of biological functions in mammalian cells by interacting with different subtypes of opiate receptors located on the plasma membrane and in the nucleus. Here we report on the identification of a new peptide transport system in the human retinal pigment epithelial (RPE) cells that transports a variety of endogenous opioid peptides with high affinity. We identified this novel, hitherto unrecognized, transport system when we were analysing the differential effects of Tat, the transacting factor encoded by HIV-1, on various transport processes in RPE cells. This transport system is markedly induced by Tat. This opioid transport system is energized by transmembrane Na+ and Cl- gradients and is distinct from any of the previously identified transport systems for opioid peptides in mammalian cells. Free amino acids, dipeptides, tripeptides and non-peptide opiate receptor antagonists are excluded by this newly identified transport system. The affinities of endogenous opioid peptides for this system are in the range of 0.4-40 microM. The identification of the high-affinity Na+- and Cl--coupled transport system in mammalian cells that is specific for endogenous opioid peptides and is induced by HIV-1 Tat is of significance not only to the biology of opioid peptides but also to the pathology of HIV-1 infection in humans.
We characterized the electrophysiology, kinetics, and quantitative structure-activity relationship (QSAR) of the human concentrative nucleoside transporter 3 (hCNT3) expressed in Xenopus laevis oocytes by measuring substrate-induced inward currents using a two-microelectrode voltage-clamp system. At membrane potentials between Ϫ30 and Ϫ150 mV, sodium activation of gemcitabine transport was sigmoidal, with a K 0.5 of 8.5 Ϯ 0.3 mM for Na ϩ and a Hill coefficient of 2.2 Ϯ 0.25 independent of membrane potential. We measured the I max and K 0.5 for substrate at Ϫ50 mV for the nucleoside analog drugs gemcitabine (638 Ϯ 58 nA, 59.7 Ϯ 17.5 M), ribavirin (546 Ϯ 37 nA, 61.0 Ϯ 13.2 M), AZT (420 Ϯ 4 nA, 310 Ϯ 9 M), and 3-deazauridine (506 Ϯ 30 nA, 50.8 Ϯ 9.90 M). K 0.5 and I max for substrate were dependent on membrane potential (both increasing as the membrane became more hyperpolarized) for all four drugs. hCNT3 also exhibited pre-steady-state currents. The quantitative structure-activity relationship (QSAR) was examined using comparative molecular field analysis and comparative molecular similarity indices analysis of the inward currents induced by 27 nucleoside analogs with substitutions at both the ribose and the nucleobase. Two statistically significant QSAR models identified electrostatic interaction as the major force in hCNT3 transport and attributed a critical role to the 3Ј-hydroxyl position of hCNT3 substrates. Steric hindrance at the 3-position and positive charge at the 5-position of the pyrimidine ring were favorable for transport. Two hCNT3 pharmacophore models revealed the minimal features required for hCNT3 transport as two hydrogen bond acceptors at 3Ј-OH and 5Ј-O and the hydrophobic center occupied by the base ring.
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