Abstract
The kinetic characteristics of [3H]adenosine uptake, the extent to which accumulated [3H]adenosine was metabolized, the effects such metabolism had on measurements of apparent Michaelis-Menten kinetic values of KT and V(max), and the sensitivities with which nucleoside transport inhibitors blocked [3H]adenosine accumulations were determined in cultured human fetal astrocytes. KT and V(max) values for accumulations of [3H]labeled purines using 15-s incubations in the absence of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and the adenosine kinase inhibitor 5'-iodotubercidin (ITU) were 6.2 μM and 0.15 nmol/min/mg of protein for the high-affinity and 2.6 mM and 21 nmol/min/mg of protein for the low-affinity components, respectively. In the presence of EHNA and ITU, where <4% of accumulated [3H]adenosine was metabolized, transport per se was measured, and kinetic values for K(T) and V(max) were 179 μM and 5.2 nmol/min/mg of protein, respectively. In the absence of EHNA and ITU, accumulated [3H]adenosine was rapidly metabolized to AMP, ADP, and ATP, and caused an appearance of 'concentrative' uptake in that the intracellular levels of [3H]-labeled purines (adenosine plus its metabolites) were 1.4- fold higher than in the medium. No apparent concentrative accumulations of [3H] adenosine were found when assays were conducted using short incubation times in the absence or presence of EHNA and ITU. The nucleoside transport inhibitors dipyridamole (DPR), nitrobenzylthioinosine (NBI), and dilazep biphasically inhibited [3H]-adenosine transport; for the inhibitor-sensitive components the IC50 values were 0.7 nM for NBI, 1.3 nM for DPR, and 3.3 nM for dilazep, and for the inhibitor-resistant component the IC50 values were 2.5 μM for NBI, 5.1 μM for dilazep, and 39.0 μM for DPR. These findings, in cultured human fetal astrocytes, represent the first demonstration of inhibitor-sensitive and -resistant adenosine transporters in nontransformed human cells.
Original language | English (US) |
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Pages (from-to) | 972-977 |
Number of pages | 6 |
Journal | Journal of Neurochemistry |
Volume | 67 |
Issue number | 3 |
DOIs | |
State | Published - Sep 1996 |
Externally published | Yes |
Keywords
- Adenosine metabolism
- Dilazep
- Dipyridamole
- Nitrobenzylthioinosine
- Nucleoside transport
ASJC Scopus subject areas
- Biochemistry
- Cellular and Molecular Neuroscience