Elsevier

Biochemical Pharmacology

Volume 59, Issue 5, 1 March 2000, Pages 477-483
Biochemical Pharmacology

Molecular and Cellular Pharmacology
Stimulation of nucleoside efflux and inhibition of adenosine kinase by A1 adenosine receptor activation

https://doi.org/10.1016/S0006-2952(99)00350-0Get rights and content

Abstract

Adenosine is produced intracellularly during conditions of metabolic stress and is an endogenous agonist for four subtypes of G-protein linked receptors. Nucleoside transporters are membrane-bound carrier proteins that transfer adenosine, and other nucleosides, across biological membranes. We investigated whether adenosine receptor activation could modulate transporter-mediated adenosine efflux from metabolically stressed cells. DDT1 MF-2 smooth muscle cells were incubated with 10 μM [3H]adenine to label adenine nucleotide pools. Metabolic stress with the glycolytic inhibitor iodoacetic acid (IAA, 5 mM) increased tritium release by 63% (P < 0.01), relative to cells treated with buffer alone. The IAA-induced increase was blocked by the nucleoside transport inhibitor nitrobenzylthioinosine (1 μM), indicating that the increased tritium release was primarily a purine nucleoside. HPLC verified this to be [3H]adenosine. The adenosine A1 receptor selective agonist N6-cyclohexyladenosine (CHA, 300 nM) increased the release of [3H]purine nucleoside induced by IAA treatment by 39% (P < 0.05). This increase was blocked by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (10 μM). Treatment of cells with UTP (100 μM), histamine (100 μM), or phorbol-12-myristate-13-acetate (PMA, 10 μM) also increased [3H]purine nucleoside release. The protein kinase C inhibitor chelerythrine chloride (500 nM) inhibited the increase in [3H]purine nucleoside efflux induced by CHA or PMA treatment. The adenosine kinase activity of cells treated with CHA or PMA was found to be decreased significantly compared with buffer-treated cells. These data indicated that adenosine A1 receptor activation increased nucleoside efflux from metabolically stressed DDT1 MF-2 cells by a PKC-dependent inhibition of adenosine kinase activity.

Section snippets

Materials

[3H]Adenine was purchased from NEN Life Sciences. PMA, adenine, adenosine, UTP, histamine, and IAA were purchased from the Sigma Chemical Co. CGS 21680 (2-[p-(2-carboxyethyl) phenylethylamino]-5′-N-ethylcarboxamidoadenosine), DPCPX, NECA, CHA, and NBMPR were purchased from Research Biochemicals International. Chelerythrine chloride was purchased from Calbiochem. Dulbecco’s modified Eagle’s medium and fetal bovine serum were obtained from Gibco BRL.

Cell culture

DDT1 MF-2 smooth muscle cells were obtained

Results

DDT1 MF-2 smooth muscle cells, loaded with [3H]adenine, were subjected to glycolytic inhibition with IAA (5 mM) (Fig. 1). IAA increased tritium efflux by 63% (P < 0.01) over release into buffer alone. The es transport inhibitor NBMPR (1 μM) was used to determine whether the increase in tritium release was due to increased efflux of [3H]purine nucleosides. NBMPR significantly blocked the IAA-induced increase (P < 0.05). As both adenosine and its metabolite, inosine, are permeants of es

Discussion

The main findings of this study were that adenosine A1 receptor activation increased the efflux of [3H]purines from DDT1 MF-2 cells during glycolytic inhibition. Stimulation of PLC by histamine H1 and P2Y nucleotide receptor activation as well as direct PKC activation with PMA mimicked this increase in efflux. Adenosine kinase assays showed that CHA or PMA treatment inhibited adenosine metabolism, an effect that could elevate intracellular adenosine levels and enhance adenosine efflux.

Depleting

Acknowledgements

This work was supported by the Medical Research Council (MRC) of Canada. F. E. P. is an MRC/RPP Investigator. C. J. D. S. is the recipient of a studentship award from the Manitoba Health Research Council and the Natural Sciences and Engineering Council of Canada. P. N. S. is the recipient of a studentship award from the MRC.

References (41)

  • R.P. Sen et al.

    Effect of forskolin and cyclic AMP analog on adenosine transport in cultured chromaffin cells

    Neurochem Int

    (1990)
  • T.D. White

    Potentiation of excitatory amino acid-evoked adenosine release from rat cortex by inhibitors of adenosine kinase and adenosine deaminase and by acadesine

    Eur J Pharmacol

    (1996)
  • K. Golembiowska et al.

    Adenosine kinase inhibitors augment release of adenosine from spinal cord slices

    Eur J Pharmacol

    (1996)
  • G.J. Keil et al.

    Altered sensory behaviors in mice following manipulation of endogenous spinal adenosine neurotransmission

    Eur J Pharmacol

    (1996)
  • J. Sawynok et al.

    Peripheral antinociceptive effect of an adenosine kinase inhibitor, with augmentation by an adenosine deaminase inhibitor, in the rat formalin test

    Pain

    (1998)
  • J.W. Assender et al.

    Expression of protein kinase C isoforms in smooth muscle cells in various states of differentiation

    FEBS Lett

    (1994)
  • T. McNally et al.

    Cloning and expression of the adenosine kinase gene from rat and human tissues

    Biochem Biophys Res Commun

    (1997)
  • S.L. Borgland et al.

    Uptake and release of [3H]formycin B via sodium-dependent nucleoside transporters in mouse leukemic L1210/MA27.1 cells

    J Pharmacol Exp Ther

    (1997)
  • J. Liu et al.

    Regulation of glutamate efflux by excitatory amino acid receptorsEvidence for tonic inhibitory and phasic excitatory regulation

    J Pharmacol Exp Ther

    (1995)
  • A. Gebauer et al.

    Modulation by 5-HT3 and 5-HT4 receptors of the release of 5-hydroxytryptamine from the guinea-pig small intestine

    Naunyn Schmiedebergs Arch Pharmacol

    (1993)
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