Stimulation of Expression for the Adenosine A2A Receptor Gene by Hypoxia in PC12 Cells: A POTENTIAL ROLE IN CELL PROTECTION

Shuichi Kobayashi,David E Millhorn

doi:10.1074/jbc.274.29.20358

Shuichi Kobayashi, David E Millhorn

Open Access

https://doi.org/10.1074/jbc.274.29.20358

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Abstract

The purpose of this study was to examine the regulation of adenosine A2A receptor (A2AR) gene expression during hypoxia in pheochromocytoma (PC12) cells. Northern blot analysis revealed that the A2AR mRNA level was substantially increased after a 3-h exposure to hypoxia (5% O2), which reached a peak at 12 h. Immunoblot analysis showed that the A2AR protein level was also increased during hypoxia. Inhibition of de novo protein synthesis blocked A2AR induction by hypoxia. In addition, removal of extracellular free Ca2+, chelation of intracellular free Ca2+, and pretreatment with protein kinase C inhibitors prevented A2AR induction by hypoxia. Moreover, depletion of protein kinase C activity by prolonged treatment with phorbol 12-myristate 13-acetate significantly inhibited the hypoxic induction of A2AR. A2AR antagonists led to a significant enhancement of A2AR mRNA levels during hypoxia, whereas A2AR agonists caused down-regulation of A2AR expression during hypoxia. This suggests that A2AR regulates its own expression during hypoxia by feedback mechanisms. We further found that activation of A2AR enhances cell viability during hypoxia and also inhibits vascular endothelial growth factor expression in PC12 cells. Thus, increased expression of A2AR during hypoxia might protect cells against hypoxia and may act to inhibit hypoxia-induced angiogenic activity mediated by vascular endothelial growth factor.

Highlights

The purpose of this study was to examine the regulation of adenosine A2A receptor (A2AR) gene expression during hypoxia in pheochromocytoma (PC12) cells
Effect of Hypoxia on Expression of Ado A2A Receptor mRNA and Protein in PC12 Cells—Northern blot analyses were performed to determine the effect of hypoxia on A2AR gene expression in PC12 cells
These results show clearly that A2AR gene expression is stimulated in a time- and dose-dependent manner by hypoxia in PC12 cells

Summary

Introduction

The purpose of this study was to examine the regulation of adenosine A2A receptor (A2AR) gene expression during hypoxia in pheochromocytoma (PC12) cells. Increased expression of A2AR during hypoxia might protect cells against hypoxia and may act to inhibit hypoxia-induced angiogenic activity mediated by vascular endothelial growth factor. The observation that A2 receptors are concentrated in brain regions that are rich in dopamine-containing cells [9, 10] suggests that the A2 receptors are involved in regulating the activity of these cells during hypoxic stress To test this possibility, we studied the effect of A2 receptor stimulation on membrane excitability in the dopaminergic pheochromocytoma (PC12) cell line [11]. An important finding was that activation of A2AR increased cell viability during exposure to 1% O2 and that activation of A2AR inhibited expression of vascular endothelial growth factor (VEGF)

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