Adenosine transport and nitrobenzylthioinosine binding in human placental membrane vesicles from brush-border and basal sides of the trophoblast.
The nucleoside transport activity of human placental syncytiotrophoblast brush-border and basal membrane vesicles was compared. Adenosine and uridine were taken up into an osmotically active space. Adenosine was rapidly metabolized to inosine, metabolism was blocked by preincubating vesicles with 2'-deoxycoformycin, and subsequent adenosine uptake studies were performed in the presence of 2'-deoxycoformycin. Adenosine influx by brush-border membrane vesicles was fitted to a two-component system consisting of a saturable system with apparent Michaelis-Menten kinetics (apparent Km approx. 150 microM) and a linear component. Adenosine uptake by the saturable system was blocked by nitrobenzylthioinosine (NBMPR), dilazep, dipyridamole and other nucleosides. Inhibition by NBMPR was associated with high-affinity binding of NBMPR to the brush-border membrane vesicles (apparent Kd 0.98 +/- 0.21 nM). Binding of NBMPR to these sites was blocked by adenosine, inosine, uridine, thymidine, dilazep and dipyridamole, and the respective apparent Ki values were 0.23 +/- 0.012, 0.36 +/- 0.035, 0.78 +/- 0.1, 0.70 +/- 0.12 (mM), and 0.12 and 4.2 +/- 1.4 (nM). In contrast, adenosine influx by basal membrane vesicles was low (less than 10% of the rate observed with brush-border membrane vesicles under similar conditions), and hence no quantitative studies of adenosine uptake could be performed with these vesicles. Nevertheless, high-affinity NBMPR binding sites were demonstrated in basal membrane vesicles with similar properties to those in brush-border membrane vesicles (apparent Kd 1.05 +/- 0.13 nM and apparent Ki values for adenosine, inosine, uridine, thymidine, dilazep and dipyridamole of 0.14 +/- 0.045, 0.54 +/- 0.046, 1.26 +/- 0.20, 1.09 +/- 0.18 mM and 0.14 and 3.7 +/- 0.5 nM, respectively). Exposure of both membrane vesicles to UV light in the presence of [3H]NBMPR resulted in covalent labeling of a membrane protein(s) with a broad apparent Mr on SDS gel electropherograms of 77,000-45,000, similar to that previously reported for many other tissues, including human erythrocytes. We conclude that the maternal (brush-border) and fetal (basal) surfaces of the human placental syncytiotrophoblast possess broad-specificity, facilitated-diffusion, NBMPR-sensitive nucleoside transporters.
Barros
L F
LF
Biomedical Sciences Division, King's College London, United Kingdom.
Bustamante
J C
JC
Yudilevich
D L
DL
Jarvis
S M
SM
D000241:Adenosine / Q000378:metabolism*; D000345:Affinity Labels; D001485:Basement Membrane / Q000378:metabolism; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008871:Microvilli / Q000378:metabolism; D009705:Nucleosides / Q000378:metabolism; D009995:Osmosis; D010920:Placenta / Q000378:metabolism* / Q000648:ultrastructure; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000378:metabolism; D014327:Trophoblasts / Q000378:metabolism*
D000345:Affinity Labels; D009705:Nucleosides; D013868:Thioinosine; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; C001789:4-nitrobenzylthioinosine; D000241:Adenosine