Electrophysiological characterization of the human Na(+)/nucleoside cotransporter 1 (hCNT1) and role of adenosine on hCNT1 function.

We previously reported that the human Na(+)/nucleoside transporter pyrimidine-preferring 1 (hCNT1) is electrogenic and transports gemcitabine and 5'-deoxy-5-fluorouridine, a precursor of the active drug 5-fluorouracil. Nevertheless, a complete electrophysiological characterization of the basic properties of hCNT1-mediated translocation has not been performed yet, and the exact role of adenosine in hCNT1 function has not been addressed either. In the present work we have used the two-electrode voltage clamp technique to investigate hCNT1 transport mechanism and study the kinetic properties of adenosine as an inhibitor of hCNT1. We show that hCNT1 exhibits presteady-state currents that disappear upon the addition of adenosine or uridine. Adenosine, a purine nucleoside described as a substrate of the pyrimidine-preferring transporters, is not a substrate of hCNT1 but a high affinity blocker able to inhibit uridine-induced inward currents, the Na(+)-leak currents, and the presteady-state currents, with a K(i) of 6.5 microM. The kinetic parameters for uridine, gemcitabine, and 5'-deoxy-5-fluorouridine were studied as a function of membrane potential; at -50 mV, K(0.5) was 37, 18, and 245 microM, respectively, and remained voltage-independent. I(max) for gemcitabine was voltage-independent and accounts for approximately 40% that for uridine at -50 mV. Maximal current for 5'-DFUR was voltage-dependent and was approximately 150% that for uridine at all membrane potentials. K(0.5)(Na(+)) for Na(+) was voltage-independent at hyperpolarized membrane potentials (1.2 mM at -50 mV), whereas I(max)(Na(+)) was voltage-dependent, increasing 2-fold from -50 to -150 mV. Direct measurements of (3)H-nucleoside or (22)Na fluxes with the charge-associated revealed a ratio of two positive inward charges per nucleoside and one Na(+) per positive inward charge, suggesting a stoichiometry of two Na(+)/nucleoside

Characterization of the rat Na+/nucleoside cotransporter 2 and transport of nucleoside-derived drugs using electrophysiological methods.

The Na(+)-dependent nucleoside transporter 2 (CNT2) mediates active transport of purine nucleosides and uridine as well as therapeutic nucleoside analogs. We used the two-electrode voltage-clamp technique to investigate rat CNT2 (rCNT2) transport mechanism and study the interaction of nucleoside-derived drugs with the transporter expressed in Xenopus laevis oocytes. The kinetic parameters for sodium, natural nucleosides, and nucleoside derivatives were obtained as a function of membrane potential. For natural substrates, apparent affinity (K(0.5)) was in the low micromolar range (12-34) and was voltage independent for hyperpolarizing membrane potentials, whereas maximal current (I(max)) was voltage dependent. Uridine and 2'-deoxyuridine analogs modified at the 5-position were substrates of rCNT2. Lack of the 2'-hydroxyl group decreased affinity but increased I(max). Increase in the size and decrease in the electronegativity of the residue at the 5-position affected the interaction with the transporter by decreasing both affinity and I(max). Fludarabine and formycin B were also transported with higher I(max) than uridine and moderate affinity (102 +/- 10 and 66 +/- 6 microM, respectively). Analysis of the pre-steady-state currents revealed a half-maximal activation voltage of about -39 mV and a valence of about -0.8. K(0.5) for Na(+) was 2.3 mM at -50 mV and decreased at hyperpolarizing membrane potentials. The Hill coefficient was 1 at all voltages. Direct measurements of radiolabeled nucleoside fluxes with the charge associated showed a ratio of two positive inward charges per nucleoside, suggesting a stoichiometry of two Na(+) per nucleoside. This discrepancy in the number of Na(+) molecules that bind rCNT2 may indicate a low degree of cooperativity between the Na(+) binding sites

Role of the human concentrative nucleoside transporter (hCNT1) in the cytotoxic action of 5[Prime]-deoxy-5-fluorouridine, an active intermediate metabolite of capecitabine, a novel oral anticancer drug.

We attempt to identify the plasma membrane transporter involved in the uptake of 5'-deoxy-5-fluorouridine (5'-DFUR), an intermediate metabolite of capecitabine. This novel oral fluoropyrimidine is used in cancer treatments and is a direct precursor of the cytostatic agent 5'-fluorouracil. We also examine the role of the transporter in 5'-DFUR cytotoxicity. The human concentrative nucleoside transporter (hCNT1) was cloned from human fetal liver and expressed in Xenopus laevis oocytes. The two-electrode voltage-clamp technique was used to demonstrate that 5'-DFUR, but not capecitabine or 5'-FU, is an hCNT1 substrate. Then, hCNT1 was heterologously expressed in the mammalian cell line Chinese hamster ovary-K1. Functional expression was demonstrated by monitoring transport of radiolabeled substrates and by using a monospecific polyclonal antibody generated against the transporter. hCNT1-expressing cells were more sensitive to 5'-DFUR than vector-transfected or wild-type cells. The sensitivity of the three cell types to other agents such as cisplatin or 5'-FU was identical. In conclusion, this study shows that 1) the pharmacological profile of a nucleoside transporter can be determined by an electrophysiological approach; 2) the hCNT1 transporter is involved in 5'-DFUR uptake; and 3) hCNT1 expression may increase cell sensitivity to 5'-DFUR treatment. This study also reports for the first time the generation of an antibody against hCNT1, which may be useful in the elucidation of the relationship between hCNT1 expression and tumor response to capecitabine treatmen

Interaction of nucleoside inhibitors of HIV-1 reverse transcriptase with the concentrative nucleoside transporter-1 (SLC28A1)

Human concentrative nucleoside transporter-1 (hCNT1) (SLC28A1) is a widely expressed, high-affinity, pyrimidine-preferring, nucleoside transporter implicated in the uptake of naturally occurring pyrimidine nucleosides as well as a variety of derivatives used in anticancer treatment. Its putative role in the uptake of other pyrimidine nucleoside analogues with antiviral properties has not been studied in detail to date. Here, using a hCNT1 stably transfected cell line and the two-electrode voltage-clamp technique, we have assessed the interaction of selected pyrimidine-based antiviral drugs, inhibitors of HIV-1 reverse transcriptase such as zidovudine (AZT), stavudine (d4T), lamivudine (3TC) and zalcitabine (ddC), with hCNT1. hCNT1 transports AZT and d4T with low affinity, whereas 3TC and ddC are not translocated, the latter being able to bind the transporter protein. Selectivity appears to rely mostly upon the presence of a hydroxyl group in the 3'-position of the ribose ring. Thus, hCNT1 cannot be considered a broad-selectivity pyrimidine nucleoside carrier; in fact, very slight changes in substrate structure provoke a dramatic shift in selectivity

Electrophysiological characterization of the human Na(+)/nucleoside cotransporter 1 (hCNT1) and role of adenosine on hCNT1 function.

We previously reported that the human Na(+)/nucleoside transporter pyrimidine-preferring 1 (hCNT1) is electrogenic and transports gemcitabine and 5'-deoxy-5-fluorouridine, a precursor of the active drug 5-fluorouracil. Nevertheless, a complete electrophysiological characterization of the basic properties of hCNT1-mediated translocation has not been performed yet, and the exact role of adenosine in hCNT1 function has not been addressed either. In the present work we have used the two-electrode voltage clamp technique to investigate hCNT1 transport mechanism and study the kinetic properties of adenosine as an inhibitor of hCNT1. We show that hCNT1 exhibits presteady-state currents that disappear upon the addition of adenosine or uridine. Adenosine, a purine nucleoside described as a substrate of the pyrimidine-preferring transporters, is not a substrate of hCNT1 but a high affinity blocker able to inhibit uridine-induced inward currents, the Na(+)-leak currents, and the presteady-state currents, with a K(i) of 6.5 microM. The kinetic parameters for uridine, gemcitabine, and 5'-deoxy-5-fluorouridine were studied as a function of membrane potential; at -50 mV, K(0.5) was 37, 18, and 245 microM, respectively, and remained voltage-independent. I(max) for gemcitabine was voltage-independent and accounts for approximately 40% that for uridine at -50 mV. Maximal current for 5'-DFUR was voltage-dependent and was approximately 150% that for uridine at all membrane potentials. K(0.5)(Na(+)) for Na(+) was voltage-independent at hyperpolarized membrane potentials (1.2 mM at -50 mV), whereas I(max)(Na(+)) was voltage-dependent, increasing 2-fold from -50 to -150 mV. Direct measurements of (3)H-nucleoside or (22)Na fluxes with the charge-associated revealed a ratio of two positive inward charges per nucleoside and one Na(+) per positive inward charge, suggesting a stoichiometry of two Na(+)/nucleoside

Adrenomedullin, a novel target for neurodegenerative diseases

Neurodegenerative diseases represent a heterogeneous group of disorders whose common characteristic is the progressive degeneration of neuronal structure and function. Although much knowledge has been accumulated on the pathophysiology of neurodegenerative diseases over the years, more efforts are needed to understand the processes that underlie these diseases and hence to propose new treatments. Adrenomedullin (AM) is a multifunctional peptide involved in vasodilation, hormone secretion, antimicrobial defense, cellular growth, and angiogenesis. In neurons, AM and related peptides are associated with some structural and functional cytoskeletal proteins that interfere with microtubule dynamics. Furthermore, AM may intervene in neuronal dysfunction through other mechanisms such as immune and inflammatory response, apoptosis, or calcium dyshomeostasis. Alterations in AM expression have been described in neurodegenerative processes such as Alzheimer’s disease or vascular dementia. This review addresses the current state of knowledge on AM and its possible implication in neurodegenerative diseases

Reduced adrenomedullin parallels microtubule dismantlement in frontotemporal lobar degeneration

Tau is a microtubule-associated protein highly expressed in neurons with a chief role in microtubule dynamics and axonal maintenance. Adrenomedullin gene (ADM) codifies for various peptides that exert broad range of actions in the body. Previous works in our groups have shown that increased ADM products are positively correlated to microtubule disruption and tau pathology in Alzheimer's disease brains. In the present study, we explore the involvement of ADM in the neuropathology of frontotemporal lobar degeneration that presents with primary tauopathy (FTLD-tau). Proteins from frontal cortices of FTLD-tau patients and age- and sex-matched non-demented controls were analyzed with antibodies against different microtubule components, including adrenomedullin, and synaptic markers. Tau pathology in frontal cortex from FTLD patients was confirmed. Levels of total βIII-tubulin as well as acetylated and detyrosinated tubulins, two markers of stabilized and aged microtubules, were significantly reduced and directly correlated with PSD95 and proBDNF in FTLD-tau patients when compared to non-demented controls. In contrast, no change in actin cytoskeleton was found. Interestingly, changes in microtubule elements, indicators of disturbed axonal preservation, were accompanied by decreased levels of free adrenomedullin, although no association was found. Altogether, reduced levels of adrenomedullin might not be directly linked to the microtubule pathology of FTLD-tau, but based on previous works, it is suggested that downregulation of ADM might be an adaptive attempt of neurons to mitigate microtubule disruption

Interaction of nucleoside inhibitors of HIV-1 reverse transcriptase with the concentrative nucleoside transporter-1 (SLC28A1)

Human concentrative nucleoside transporter-1 (hCNT1) (SLC28A1) is a widely expressed, high-affinity, pyrimidine-preferring, nucleoside transporter implicated in the uptake of naturally occurring pyrimidine nucleosides as well as a variety of derivatives used in anticancer treatment. Its putative role in the uptake of other pyrimidine nucleoside analogues with antiviral properties has not been studied in detail to date. Here, using a hCNT1 stably transfected cell line and the two-electrode voltage-clamp technique, we have assessed the interaction of selected pyrimidine-based antiviral drugs, inhibitors of HIV-1 reverse transcriptase such as zidovudine (AZT), stavudine (d4T), lamivudine (3TC) and zalcitabine (ddC), with hCNT1. hCNT1 transports AZT and d4T with low affinity, whereas 3TC and ddC are not translocated, the latter being able to bind the transporter protein. Selectivity appears to rely mostly upon the presence of a hydroxyl group in the 3'-position of the ribose ring. Thus, hCNT1 cannot be considered a broad-selectivity pyrimidine nucleoside carrier; in fact, very slight changes in substrate structure provoke a dramatic shift in selectivity

Role of the human concentrative nucleoside transporter (hCNT1) in the cytotoxic action of 5[Prime]-deoxy-5-fluorouridine, an active intermediate metabolite of capecitabine, a novel oral anticancer drug.

We attempt to identify the plasma membrane transporter involved in the uptake of 5'-deoxy-5-fluorouridine (5'-DFUR), an intermediate metabolite of capecitabine. This novel oral fluoropyrimidine is used in cancer treatments and is a direct precursor of the cytostatic agent 5'-fluorouracil. We also examine the role of the transporter in 5'-DFUR cytotoxicity. The human concentrative nucleoside transporter (hCNT1) was cloned from human fetal liver and expressed in Xenopus laevis oocytes. The two-electrode voltage-clamp technique was used to demonstrate that 5'-DFUR, but not capecitabine or 5'-FU, is an hCNT1 substrate. Then, hCNT1 was heterologously expressed in the mammalian cell line Chinese hamster ovary-K1. Functional expression was demonstrated by monitoring transport of radiolabeled substrates and by using a monospecific polyclonal antibody generated against the transporter. hCNT1-expressing cells were more sensitive to 5'-DFUR than vector-transfected or wild-type cells. The sensitivity of the three cell types to other agents such as cisplatin or 5'-FU was identical. In conclusion, this study shows that 1) the pharmacological profile of a nucleoside transporter can be determined by an electrophysiological approach; 2) the hCNT1 transporter is involved in 5'-DFUR uptake; and 3) hCNT1 expression may increase cell sensitivity to 5'-DFUR treatment. This study also reports for the first time the generation of an antibody against hCNT1, which may be useful in the elucidation of the relationship between hCNT1 expression and tumor response to capecitabine treatmen

Increased levels of brain adrenomedullin in the neuropathology of Alzheimer's Disease

Alzheimer's disease (AD) is characterized by the loss of synaptic contacts caused in part by cytoskeleton disruption. Adrenomedullin (AM) is involved in physiological functions such as vasodilation, hormone secretion, antimicrobial activity, cellular growth, and angiogenesis. In neurons, AM and related peptides are associated with some structural and functional cytoskeletal proteins, causing microtubule destabilization. Here, we describe the relationships between AM and other signs of AD in clinical specimens. Frontal cortex from AD patients and controls were studied for AM, acetylated tubulin, NCAM, Ox-42, and neurotransmitters. AM was increased in AD compared with controls, while levels of acetylated tubulin, NCAM, and neurotransmitters were decreased. Interestingly, increases in AM statistically correlated with the decrease in these markers. Furthermore, Ox42 overexpression in AD correlated with levels of AM. It is proposed that AD patients may have neural cytoskeleton failure associated with increase of AM levels, resulting in axon transport collapse and synaptic loss. These observations suggest that reducing AM expression may constitute a new avenue to prevent/treat AD