Affinity of calcium channel inhibitors, benzodiazepines, and other vasoactive compounds for the nucleoside transport system

1985 ◽  
Vol 63 (10) ◽  
pp. 1302-1307 ◽  
Author(s):  
James R. Hammond ◽  
Evan F. Williams ◽  
Alexander S. Clanachan
Keyword(s):  
Guinea Pig ◽  
Calcium Channel ◽  
Transport System ◽  
Human Erythrocytes ◽  
Nucleoside Transport ◽  
Rat Tissues ◽  
High Affinity ◽  
Cardiac Membranes ◽  
Transport Inhibitors ◽  
Calcium Channel Inhibitors

There is evidence to suggest that several different groups of drugs including the so-called coronary vasodilators, benzodiazepines, and calcium channel inhibitors may owe their vasoactivity, in part, to the potentiation of the vasorelaxant effects of endogenous adenosine. To measure the affinity of some of these agents for the membrane-located nucleoside transport system, competition binding assays have been performed using the high-affinity radioligand [3H]nitrobenzylthioinosine (NBMPR). Experiments were performed on human erythrocytes and cardiac membranes from guinea pigs and rats. Recognized nucleoside transport inhibitors had high affinity (<50 nM) for NBMPR recognition sites associated with the nucleoside transporter complex in human erythrocytes, whereas calcium channel inhibitors and benzodiazepines had predominantly low affinity (> 1 μM). Although some recognized transport inhibitors, such as dipyridamole, show marked differences in affinity for NBMPR sites in guinea pig and rat tissues, benzodiazepines and calcium channel blockers displayed no such species selectivity and had low affinity (> 1 μM) for NBMPR sites in both guinea pig and rat cardiac membranes. Consequently, it is unlikely that agents such as benzodiazepines and calcium channel inhibitors cause significant inhibition of adenosine transport, and hence potentiate adenosine actions, at the concentrations required to induce effects through occupation of their respective, specific high-affinity sites.

scholarly journals [3H]dipyridamole binding to nucleoside transporters from guinea-pig and rat lung

1986 ◽  
Vol 240 (3) ◽  
pp. 879-883 ◽  
Author(s):  
M M Shi ◽  
J D Young
Keyword(s):  
Guinea Pig ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Affinity Binding ◽  
Nucleoside Transporter ◽  
High Affinity Binding ◽  
High Affinity ◽  
Nucleoside Transport Inhibitors ◽  
Transport Inhibitors ◽  
Low Sensitivity

Membranes from guinea-pig lung exhibited high-affinity binding of [3H]dipyridamole, a potent inhibitor of nucleoside transport. Binding (apparent KD 2 nM) was inhibited by the nucleoside-transport inhibitors nitrobenzylthioinosine (NBMPR), dilazep and lidoflazine and by the transported nucleosides uridine and adenosine. In contrast, there was no detectable high-affinity binding of [3H]dipyridamole to lung membranes from the rat, a species whose nucleoside transporters exhibit a low sensitivity to dipyridamole inhibition. Bmax. values for high-affinity binding of [3H]dipyridamole and [3H]NBMPR to guinea-pig membranes were similar, suggesting that these structurally unrelated ligands bind to the NBMPR-sensitive nucleoside transporter with the same stoichiometry.

Nucleoside transport in heart: species differences in nitrobenzylthioinosine binding, adenosine accumulation, and drug-induced potentiation of adenosine action

1984 ◽  
Vol 62 (1) ◽  
pp. 31-37 ◽  
Author(s):  
Evan F. Williams ◽  
Philip H. Barker ◽  
A. S. Clanachan
Keyword(s):  
Guinea Pig ◽  
Rat Heart ◽  
Binding Capacity ◽  
Specific Binding ◽  
Nucleoside Transport ◽  
Inotropic Action ◽  
Guinea Pig Heart ◽  
Cardiac Membranes ◽  
Nucleoside Transport Inhibitors ◽  
Transport Inhibitors

The site-specific binding of the potent and selective nucleoside transport inhibitor, [3H]nitrobenzylthioinosine (NBMPR), to the nucleoside transport system of cardiac membranes of several species was investigated. The affinity of [3H]NBMPR for these sites ranged from 0.03 nM in rat to 0.78 nM in dog. The maximal binding capacity of cardiac membranes for [3H]NBMPR was also species dependent and was greatest in bovine and guinea pig heart (2551 and 1700 fmol/mg protein, respectively) and least in rat (195 fmol/mg protein). The affinities of recognized nucleoside transport inhibitors and benzodiazepines for these transport inhibitory sites in guinea pig and rat heart were estimated by studying the inhibition of the site-specific binding of [3H]NBMPR in competition experiments. These values were compared with their inhibitory effects on the transporter-dependent accumulation of [3H]adenosine in guinea pig and rat cardiac muscle segments and with their ability to potentiate the negative inotropic action of adenosine in electrically driven guinea pig and rat left atria. In guinea pig heart, the recognized nucleoside transport inhibitors and benzodiazepines had an order of affinity (dilazep > hydroxynitrobenzylthioguanosine > dipyridamole > hexobendine [Formula: see text] lidoflazine [Formula: see text] flunitrazepam > diazepam > lorazepam > flurazepam) for the NBMPR site which was similar to those for the inhibition of [3H]adenosine accumulation and for potentiation of adenosine action. In contrast, in rat heart, where the maximal binding capacity of [3H]NBMPR was lower (eightfold), the nucleoside transporter dependent accumulation of [3H]adenosine was also lower (sixfold) and the negative inotropic action of adenosine was not significantly potentiated. Furthermore, NBMPR sites in rat heart displayed a significantly lower affinity for hexobendine, dipyridamole, and lidoflazine relative to sites in guinea pig hearts. These data indicate that significant differences in cardiac nucleoside transport systems exist among species with respect to both membrane density and drug affinity. Such differences influence the ability of transport inhibitors to modify adenosine action in these tissues.

scholarly journals Nucleoside transport in human and sheep erythrocytes. Evidence that nitrobenzylthioinosine binds specifically to functional nucleoside-transport sites

1980 ◽  
Vol 190 (2) ◽  
pp. 377-383 ◽  
Author(s):  
S M Jarvis ◽  
J D Young
Keyword(s):  
Transport System ◽  
Binding Sites ◽  
Sheep Erythrocyte ◽  
Specific Interaction ◽  
Human Erythrocytes ◽  
Nucleoside Transport ◽  
Erythrocyte Membranes ◽  
Sheep Erythrocytes ◽  
High Affinity ◽  
Transport Site

Nitrobenzyl[35S]thioinosine binding and nitro[3H]benzylthioinosine binding to nucleoside-permeable and nucleoside-impermeable sheep erythrocyte membranes was investigated, and compared with that found for human erythrocytes. High-affinity nitrobenzylthioinosine-binding sites (apparent KD congruent to 1 nM) were present on human and nucleoside-permeable but not nucleoside-impermeable sheep erythrocyte membranes (8400 and 18 sites/cell for human and sheep nucleoside-permeable sheep erythrocytes was displaced by nitrobenzylthioguanosine and dipyridamole. Uridine, inosine and adenosine inhibited binding. The smaller number of nitrobenzylthioinosine sites on nucleoside-permeable cells compared with human erythrocytes corresponded to a considerably lower Vmax. for uridine influx in these cells (0.53 × 10(-20) mol/cell per s at 25 degrees C compared with 254 × 10(-20) mol/cell per s). It is suggested that high-affinity nitrobenzylthioinosine binding represents a specific interaction with functional nucleoside-transport sites. The uridine-translocation capacity for each transport site at 25 degrees C is 180 molecules/site per s for both nucleoside-permeable sheep cells and human erythrocytes (assuming a 1:1 interaction between nitrobenzylthioinosine and the nucleoside-transport system).

scholarly journals Species differences in nucleoside transport. A study of uridine transport and nitrobenzylthioinosine binding by mammalian erythrocytes

1982 ◽  
Vol 208 (1) ◽  
pp. 83-88 ◽  
Author(s):  
S M Jarvis ◽  
J R Hammond ◽  
A R P Paterson ◽  
A S Clanachan
Keyword(s):  
Guinea Pig ◽  
Binding Sites ◽  
Species Differences ◽  
Cell Types ◽  
Nucleoside Transport ◽  
Kinetic Properties ◽  
Affinity Binding ◽  
Transport Capacity ◽  
High Affinity Binding ◽  
High Affinity

A kinetic study of the inward transport of uridine in erythrocytes of rabbit, human, mouse, rat and guinea-pig demonstrated that the apparent Km of this process was similar (about 0.2mM) in these cell types, but Vmax. values differed markedly. In this array of cell types, Vmax. values were proportional to the number of transport-inhibitory, high-affinity binding sites present per cell of each type. Transport of uridine or adenosine was not detected in dog erythrocytes, nor was saturable, high-affinity binding of nitrobenzylthioinosine demonstrable. These findings demonstrate that species differences in nucleoside transport capacity are attributable to differences in the cell-surface content of functional nucleoside transport sites, rather than to differences in the kinetic properties of these sites.

A purine-selective nucleobase/nucleoside transporter in PK15NTD cells

2008 ◽  
Vol 294 (6) ◽  
pp. R1988-R1995 ◽  
Author(s):  
Kazi Mirajul Hoque ◽  
Linxi Chen ◽  
George P. H. Leung ◽  
Chung-Ming Tse
Keyword(s):  
Transport System ◽  
Mammalian Cells ◽  
Organic Cation ◽  
Organic Cation Transporter ◽  
Nucleoside Transport ◽  
Acidic Ph ◽  
Nucleoside Transporter ◽  
High Affinity ◽  
Naturally Occurring ◽  
Purine Analog

Nucleoside and nucleobase transporters are important for salvage of purines and pyrimidines and for transport of their analog drugs into cells. However, the pathways for nucleobase translocation in mammalian cells are not well characterized. We identified an Na-independent purine-selective nucleobase/nucleoside transport system in the nucleoside transporter-deficient PK15NTD cells. This transport system has 1,000-fold higher affinity for nucleobases than nucleosides with Kmvalues of 2.5 ± 0.7 μM for [3H]adenine, 6.4 ± 0.5 μM for [3H]guanine, 1.1 ± 0.1 mM for [3H]guanosine, and 4.2 ± 0.5 mM [3H]adenosine. The uptake of [3H]guanine (0.05 μM) was inhibited by other nucleobases and nucleobase analog drugs (at 0.5–1 mM in the order of potency): 6-mercaptopurine = thioguanine = guanine > adenine >>> thymine = fluorouracil = uracil. Cytosine and methylcytosine had no effect. Nucleoside analog drugs with modification at 2′ and/or 5 positions (all at 1 mM) were more potent than adenosine in competing the uptake of [3H]guanine: 2-chloro-2′-deoxyadenosine > 2-chloroadenosine > 2′3′-dideoxyadenosine = 2′-deoxyadenosine > 5-deoxyadenosine > adenosine. 2-Chloro-2′-deoxyadenosine and 2-chloroadenosine inhibited [3H]guanine uptake with IC50values of 68 ± 5 and 99 ± 10 μM, respectively. The nucleobase/nucleoside transporter was resistant to nitrobenzylthioinosine {6-[(4-nitrobenzyl) thiol]-9-β-d-ribofuranosylpurine}, dipyridamole, and dilazep, but was inhibited by papaverine, the organic cation transporter inhibitor decynium-22 (IC50of ∼1 μM), and by acidic pH (pH = 5.5). In conclusion, we have identified a mammalian purine-selective nucleobase/nucleoside transporter with high affinity for purine nucleobases. This transporter is potentially important for transporting naturally occurring purines and purine analog drugs into cells.

Bovine adrenal endothelial cells express nucleoside transporters nonregulated by protein kinases A and C

1996 ◽  
Vol 271 (2) ◽  
pp. C504-C510 ◽  
Author(s):  
R. P. Sen ◽  
L. Sobrevia ◽  
E. G. Delicado ◽  
D. Yudilevich ◽  
M. T. Miras-Portugal
Keyword(s):  
Endothelial Cells ◽  
Protein Kinases ◽  
Transport System ◽  
Chromaffin Cells ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Facilitated Diffusion ◽  
High Affinity ◽  
Adenosine Transport ◽  
Stimulation Of

The present investigation characterizes the nucleoside transporters in bovine adrenomedullary endothelial cells and their possible regulation by the action of protein kinases A and C to establish comparisons with the nucleoside transport system in chromaffin cells. The nucleoside transport proved to be a nitrobenzylthioinosine (NBTI)-sensitive facilitated-diffusion system with high affinity for adenosine. These endothelial cells had a high density of nucleoside transporters (660,000 +/- 130,000 transporters/ cell), measured by NBTI binding, and the efficiency was close to 2 adenosine molecules internalized transporter-1.s-1. The stimulation of the cells with bradykinin and P1,P4-di(adenosine-5')tetraphosphate, which raise the intracellular Ca2+ concentration, did not modulate the adenosine transport. When the cells were stimulated with signals coupled to adenosine 3',5'-cyclic monophosphate intracellular production, such as norepinephrine and isoproterenol, the adenosine transport was not modified. Furthermore, the treatment of the cells with direct activators of both protein kinases A and C had no effect on adenosine transport, in contrast to that reported in chromaffin cells.

Selective transport of adenosine into porcine coronary smooth muscle

2000 ◽  
Vol 279 (3) ◽  
pp. H1397-H1410 ◽  
Author(s):  
L. J. Rubin ◽  
L. R. Johnson ◽  
J. R. Dodam ◽  
A. K. Dhalla ◽  
L. Magliola ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Isolated Cells ◽  
Extracellular Adenosine ◽  
Coronary Smooth Muscle ◽  
Porcine Coronary Artery ◽  
High Affinity ◽  
Nucleoside Transport Inhibitors ◽  
Transport Inhibitors

Adenosine (ADO), an endogenous regulator of coronary vascular tone, enhances vasorelaxation in the presence of nucleoside transport inhibitors such as dipyridamole. We tested the hypothesis that coronary smooth muscle (CSM) contains a high-affinity transporter for ADO. ADO-mediated relaxation of isolated large and small porcine coronary artery rings was enhanced 12-fold and 3.4-fold, respectively, by the transport inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI). Enhanced relaxation was independent of endothelium and was selective for ADO over synthetic analogs. Uptake of [3H]ADO into freshly dissociated CSM cells or endothelium-denuded rings was linear and concentration dependent. Kinetic analysis yielded a maximum uptake ( V max) of 67 ± 7.0 pmol · mg protein−1 · min−1 and a Michaelis constant ( K m) of 10.5 ± 5.8 μM in isolated cells and a V max of 5.1 ± 0.5 pmol · min−1 · mg wet wt−1and a K m of 17.6 ± 2.6 μM in intact rings. NBTI inhibited transport into small arteries (IC50 = 42 nM) and cells. Analyses of extracellular space and diffusion kinetics using [3H]sucrose indicate the V max and K m for ADO transport are sufficient to clear a significant amount of extracellular adenosine. These data indicate CSM possess a high-affinity nucleoside transporter and that the activity of this transporter is sufficient to modulate ADO sensitivity of large and small coronary arteries.

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