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Endogenous Expression of Adenosine A1, A2 and A3 Receptors in Rat C6 Glioma Cells

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Abstract

Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [3H]DPCPX, selective A1R antagonist, revealed a single binding site with a K D = 9.4 ± 1.4 nM and B max = 62.7 ± 8.6 fmol/mg protein. Binding of [3H]DPCPX in intact cell revealed a K D = 17.7 ± 1.3 nM and B max = 567.1 ± 26.5 fmol/mg protein. On the other hand, [3H]ZM241385 binding experiments revealed a single binding site population of receptors with K D = 16.5 ± 1.3 nM and B max = 358.9 ± 52.4 fmol/mg protein in intact cells, and K D = 4.7 ± 0.6 nM and B max = 74.3 ± 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A2A receptor in C6 cells. A1, A2A, A2B and A3 adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Giα and Gsα proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A1R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A1 and A2 receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.

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Abbreviations

A1R:

Adenosine A1 receptors

A2AR:

Adenosine A2A receptors

A2BR:

Adenosine A2B receptors

A3R:

Adenosine A3 receptors

ADA:

adenosine deaminase

CGS 21680:

2-[p-(2-carboxyethyl) phenylamino]-5´-N-ethylcarboxiamido adenosine

CHA:

N6-cyclohexyladenosine

CNS:

central nervous system

CPA:

N6-cyclopentyladenosine

[3H]DPCPX:

Cyclopentyl-1,3-dypropylxanthine,8-[dipropy-2,3−3H(N)]

Gpp(NH)p:

Guanylylimido-diphosphate

G-Protein:

GTP-binding protein

NECA:

5´-(N-ethylcarboxamido) adenosine

PSB1115:

1-propyl-8-(4-sulfophenyl)xanthine

PTX:

pertussis toxin

R-PIA:

R(-)N6-(2-phenylisopropyl) adenosine

ZM241385:

(4-(2-[7-amino-2-(2-fury1)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol)

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Acknowledgments

This work has been supported by DGES grant BFI2005-00582 and JCCM grants PAI-05-043 and GC05-003. Carlos Alberto Castillo is the recipient of a predoctoral fellowship from J.C.C.M.

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  1. Departamento de Química Inorgánica, Orgánica y Bioquímica, Facultad de Químicas, Centro Regional de Investigaciones Biomédicas, Universidad de Castilla-La Mancha, Ciudad Real, Spain

    Carlos Alberto Castillo, José Luís Albasanz, Mercedes Fernández & Mairena Martín

  2. Área de Bioquímica, Facultad de Ciencias Químicas, Universidad de Castilla-La Mancha, Avenida Camilo José Cela 10, Ciudad Real, 13071, Spain

    Mairena Martín

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  1. Carlos Alberto Castillo
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  2. José Luís Albasanz
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  3. Mercedes Fernández
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Correspondence to Mairena Martín.

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Castillo, C.A., Albasanz, J.L., Fernández, M. et al. Endogenous Expression of Adenosine A1, A2 and A3 Receptors in Rat C6 Glioma Cells. Neurochem Res 32, 1056–1070 (2007). https://doi.org/10.1007/s11064-006-9273-x

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