All nucleoside transporters in bovine chromaffin cells are nitrobenzylthioinosine sensitive (original) (raw)
Related papers
Neuroscience Letters, 1993
Nitrobenzylthioinosine (NBTI) is a high affinity probe for facilitated diffusion nucleoside transporters. Kinetic analysis of the binding of[3tt]NBTI to plasma membranes ofchromaffin cells was conducted in the presence or absence of adenosine 5'-triphosphate (ATP). Similar curvilinear plots with a Hill number of 1.32 were obtained in both conditions. ATP significantly increased the number of NBTI binding sites in these preparations showing Bm,x values of 1.62 + 0.20 pmol/mg protein for controls and 3.22 +_ 0.31 pmol/mg protein in the presence of ATE However, the affinity constant (KD) was not significantly modified. The non-metabolizable ATP analogue, 5'-adenylyl imidodiphosphate (AMP-PNP) and diadenosine tetraphosphate (Ap4A) can mimic the stimulatory ATP effect, but adenosine monophosphate (AMP) has no effect on the NBTI binding to plasma membranes. These results indicate a modulatory role tbr ATE non-hydrolysis dependent, on nucleoside transport in chromaffin cells. Therefore, a nucleotide binding site on the nucleoside transporter similar to that described for glucose transporter could be suggesled.
Nucleoside transport sites in rat brain membrane preparations were labeled with [3H]nitrobenzylthioinosine ([3H]NBI), a potent inhibitor of nucleoside transport systems. The membranes contained a single class of very high affinity binding sites with K(D) and B(max) values of 0.06 nM and 147 fmol/mg of protein, respectively. The displacement of [3H]NBI binding by various nucleosides, adenosine receptor agonists and antagonists, and known nucleoside transport inhibitors was examined. The K(i) values (micromolar concentration) of [3H]NBI displacement by the nucleosides tested were: adenosine, 3.0; inosine, 160; thymidine, 240; uridine, 390; guanosine, 460; and cytidine, 1000. These nucleosides displayed parelled displacement curves indicating their interaction with a common site labeled by [3H]NBI. The nucleobases, hypoxanthine and adenine, exhibited K(i) values of 220 and 3640 μM, respectively. Adenosine receptor agonists exhibited moderate affinities for the [3H]NBI site, whereas the adenosine receptor antagonists, caffeine, theophylline, and enprofylline, were ineffective displacers. The K(i) values for cyclohexyladenosine, (+)- and (-)-phenylisopropyladenosine, 2-chloroadenosine, and adenosine 5'-ethylcarboxamide were 0.8, 0.9, 2.6, 12, and 54 μM, respectively. These affinities and the rank order of potencies indicate that [3H]NBI does not label any known class of adenosine receptors (i.e., A1, A2, and P). The K(i) values of other nucleoside transport inhibitors were; nitrobenzylthioguanosine, 0.05 nM; dipyridamole, 16 nM; papaverine, 3 μM; and 2'-deoxyadenosine, 22 μM. These results indicate that [3H]NBI binds to a nucleoside transporter in brain which specifically recognizes adenosine as its preferred endogenous substrate. This ligand may aid in the identification of CNS neural systems that selectively accumulate adenosine and thereby control 'adenosinergic' function.
Effect of 5′‐(N‐Ethylcarboxamido) adenosine on Adenosine Transport in Cultured Chromaffin Cells
Journal of …, 1990
Esmerilda G. Delicado, *Alexandra Rodrigues, Raquel P. Sen, *Ana M. Sebastiao, *J. Alexandre Ribeiro, and M. Teresa Miras-Portugal ... Departamento de Bioquhica, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain; and *Laboratory of Pharmacology, ...
Regulation of nitrobenzylthioinosine-sensitive adenosine uptake by cultured kydney cells
The American journal of physiology
The effect of nitrobenzylthioinosine (NBTI) on [3H]adenosine uptake and the characterization of the [3H]NBTI binding in cell (primary cultures and LLC-PK1 cell line) plasma membrane and brush-border membrane (BBM) vesicles from pig renal cortices and LLC-PK1 cells was analyzed. [3H]adenosine uptake was strongly inhibited by NBTI in nonconfluent cells, whereas it was totally insensitive to the reagent in BBM. The concentration dependence of [3H]adenosine uptake in BBM was linear, suggesting simple diffusion. In both cell membranes and BBM high-affinity [3H]NBTI binding was observed. [3H]NBTI binding as well as NBTI-sensitive [3H]adenosine uptake was strongly reduced when cells grew to confluence. Both reduction effects were reproduced by treatment of nonconfluent cells with chlorophenyl adenosine 3',5'-cyclic monophosphate (cAMP), which indicates that the transporter is regulated by a cAMP-dependent protein kinase. To confirm this hypothesis, the binding of [3H]NBTI was analy...
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1992
The dynamics of the nitrobenzylthioinosine (NBTI)-sensitive nucleoside transporter were studied in cultured chromaffin cells. Photolabelling of transporters with [3H]NBTI induced a down-regulation of this protein from the plasma membrane with a half-life value of 2.31 +/- 0.61 h, measured by specific isolation of plasma membrane on polycationic beads. In this internalization step 50-60% of transporters were destroyed. The remaining labelled protein reappeared in plasma membranes and underwent a new disappearance cycle with a longer half-life period (34.65 +/- 3.9 h). A similar pattern of internalization and reappearance of nucleoside transporters was observed in cells cross-linked with non-labelled NBTI, with a half value of reappearance of 33 h. Chromaffin cells cultured in the presence of the protein synthesis inhibitor, cycloheximide, had a component of disappearance for NBTI binding sites with a half-life value of 24.6 +/- 1.4 h.
European Journal of Biochemistry, 1994
Uridine transport was investigated in cultured chromaffin cells and plasma membrane vesicles from chromaffin tissue. In intact cells, the kinetic parameters for uridine uptake were K, 150 2 45 pM, and V,,, 414 2 17 pmol . lo6 cells-' . min-I. This low affinity for uridine and its inhibition by low concentrations of nitrobenzylthioinosine (K, 3 nM) and dipyridamole (K, 54 nM) are consistent with a facilitated diffusion nucleoside transport system. The IC,* value for the adenosine transport inhibition by uridine was very high (240 pM), agreeing with the relative affinities of these nucleosides in the chromaffin cell nucleoside transport system, which was 150-fold higher for adenosine than for uridine. Uridine was significantly metabolized in chromaffin cells but not in plasma membrane vesicles. The affinity of uridine transport measured in these membrane vesicles was reproducible and similar to the affinity found for intact cells with a K,,, value of 185 % 11 pM and a V,, value of 4.2420.10 pmol . mg protein-' . s-I. These membrane preparations were employed to investigate the regulatory action of ATP and other nucleotide analogues on nucleoside transport. ATP increased the V,,, value but the K, value was not significantly modified. Adenosine 5'-[p,y-imino]triphosphate, 1 ,W-ethenoadenosine 5'-triphosphate, and adenosine(5')-tetraphospho(5')adenosine (Ap,A) at 100 pM were able to mimic the ATP effect. These results agree with a regulatory role of ATP, and the uridine transport on chromaffin plasma membrane vesicles is a good model for analyzing the nucleoside-transporter function and regulation.
The Biochemical journal, 1983
Nucleoside transport in various types of animal cells is inhibited by the binding of nitrobenzylthioinosine (NBMPR) to a set of high-affinity sites on the plasma membrane. This work examined the binding of [3H]NBMPR to the nucleoside transporters of cultured Nil 8 hamster fibroblasts and of cells of a virus-transformed clone (Nil SV) derived from Nil 8. Experiments conducted with intact Nil 8 and Nil SV cells and with membrane preparations indicated that the two lines differed significantly in the cellular content of binding sites and only slightly in the affinities of these sites for NBMPR. Nil 8 and Nil SV cells possessed (4.2-8.0) X 10(5) and (2.0-4.0) X 10(6) sites per cell respectively, whereas the dissociation constants of site-bound NBMPR obtained with intact cells and with membrane preparations were similar, ranging from 0.29 to 1.5 nM. Dilazep, a potent inhibitor of nucleoside transport that is structurally unrelated to NBMPR, appeared to compete with NBMPR for binding to t...
Adenosine transport in bovine chromaffin cells in culture
The Journal of biological chemistry, 1986
Bovine adrenal chromaffin cells in culture have a high capacity and affinity for adenosine uptake with Vmax = 14 +/- 2.4 pmol/10(6) cells/min (133 pmol/mg of protein/min) and Km = 1 +/- 0.2 microM. Transport studies, at short time periods, in recently isolated chromaffin cells have Vmax = 15 pmol/10(6) cells/min and Km = 1.1 microM in ATP-depleted cells. Endogenous levels of the various purine nucleosides and bases were determined by high pressure liquid chromatography, with adenosine (3 +/- 1 nmol/10(6) cells), inosine (5.3 +/- 1.2 nmol/10(6) cells), and hypoxanthine (2.1 +/- 0.8 nmol/10(6) cells) being the purine metabolites found in the highest concentration. Taking into account the intracellular water, endogenous levels of 2.1, 3.8, and 1.5 mM, respectively, were obtained. Radioactively labeled adenosine inside the cell underwent enzymatic transformations, producing inosine, hypoxanthine, xanthine, and nucleotides, with their appearance and distribution being a function of the i...
Effect of forskolin and cyclic AMP analog on adenosine transport in cultured chromaffin cells
Neurochemistry International, 1990
The adenosine transport in cultured chromattin cells was inhibited by the presence of the adenylate cyclase activator, forskolin, and a cAMP analog. The Vmax values of this transport obtained for control and in the presence of 8-(-4-chlorophenylthio)adenosine-Y : 5'-monophosphate cyclic (C1PhcAMP, 100 aM) or forskolin (0.5/~M) were 85 + 5; 45 + 1.5 and 38 __+ 3 pmol/106 cells/rain, respectively. The Km values were not significantly modified.
American Association for Cancer Research, 1983
In studies using a rapid kinetic technique, evidence was derived for multiplicity of systems mediating [3H]adenosine transport in L1210 cells. A variety of approaches were used in discriminating between transport and kinase-mediated phosphorylation. Under these conditions, two systems mediating influx were delineated which exhibited high-affinity [Km = 13.9 ± 2 (S.E.) UM] or low-affinity [Km = 199 ± 27 /XM] for [3H]adenosine. Both systems exhibited high capacities, but that associated with the low-affinity system (V„a°x = 263 ±43 nmol/ sec/g, dry weight) was 2-to 3-fold greater than that for the high-affinity system (Vâ"¢,= 99.6 ±12 nmol sec/g, dry weight). The relative difference in affinity of these two systems during influx was also reflected in the values for influx K, obtained with other nucleosides and nucleoside analogues. Influx of [3H]adenosine by each mediated system was inhibited by 6-(2hydroxy-5-nitrobenzyl)thioguanosine, a specific transport in hibitor, and by 9-/?-D-arabinofuranosylpurine-6(1H)thione which is not phosphorylated in L1210 cells. Influx kinetics were the same in L1210 cells, in adenosine tri phosphate-depleted L1210 cells, in cells (L1210/ara-C/MMPR) which have sub stantially reduced ability for [3H]adenosine phosphorylation, and in the presence of 2'-deoxycoformycin, a potent inhibitor of adenosine deaminase. The same multiplicity in mediated influx of [3H]adenosine was shown at 0°when transport be came rate limiting to total uptake. The high-affinity system mediating [ :Hláclenosme influx was also elucidated in L1210 cell plasma membrane vesicles in the presence or absence of 2'-deoxycoformycin. Almost all of the natural nucleosides ex amined competed less effectively with [3H]adenosine for influx by the high-affinity system than by the low-affinity system. These results are discussed with respect to possible pharma cological implications.