Characterization of inhibitor-sensitive and -resistant adenosine transporters in cultured human fetal astrocytes

J. G. Gu, A. Nath, J. D. Geiger

Research output: Contribution to journalArticle

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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 languageEnglish (US)
Pages (from-to)972-977
Number of pages6
JournalJournal of Neurochemistry
Volume67
Issue number3
StatePublished - Sep 1 1996

Fingerprint

Astrocytes
Adenosine
Dilazep
Dipyridamole
Purines
Nucleosides
Inhibitory Concentration 50
Adenosine Deaminase Inhibitors
Adenosine Kinase
Enzyme kinetics
Proteins
Kinetics
adenosine transporter
Adenosine Monophosphate
Metabolites
Metabolism
Adenosine Diphosphate
Assays
Demonstrations
Adenosine Triphosphate

Keywords

  • Adenosine metabolism
  • Dilazep
  • Dipyridamole
  • Nitrobenzylthioinosine
  • Nucleoside transport

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

Characterization of inhibitor-sensitive and -resistant adenosine transporters in cultured human fetal astrocytes. / Gu, J. G.; Nath, A.; Geiger, J. D.

In: Journal of Neurochemistry, Vol. 67, No. 3, 01.09.1996, p. 972-977.

Research output: Contribution to journalArticle

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