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Signaling via A2A adenosine receptor in four PC12 cell clones
G Arslan1, B Kull, B B Fredholm
1Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden. giulia.arslan@fyfa.ki.se
Abstract:
PC12 cells are genetically labile and so-called wild-type cells comprise multiple subclones. We have examined the A2A adenosine receptor signal transduction pathways in four such clones (denoted clones 1, 19, 21 and 27) of PC12 cells. Adenosine A2A, A2B and A1 receptor mRNAs were detected in all four clones by RT-PCR, whereas no A3 receptor mRNA was found. A2A receptors were quantitated by radioligand binding using the antagonist radioligand [3H]SCH 58261 ([3H]-5-amino-7(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4 triazolo [1,5-c] pyrimidine). The Bmax was highest in clone 1 followed by clones 21, 19 and 27. Whereas the amount of G(i) protein appeared similar in all four clones, the amount of G(s) protein was higher in clones 21 and 27 than in the other two clones. Maximal responses to the non-selective adenosine analogue NECA (5'-N-ethylcarboxamidoadenosine) were similar to those observed with the selective adenosine A2A receptor agonist CGS 21680 (2-[p-(2-carbonylethyl) phenylethylamino]-5'-N-ethylcarboxamidoadenosine), and were approximately equal in clones I and 21, but lower in clone 19 and very low in clone 27. For both compounds EC50 was significantly higher in clone 27 than in clone 1. In both clones the response to NECA could be competitively antagonized by a selective adenosine A2A antagonist, SCH 58261. The present results show that different clones of PC 12 cells differ widely in the cAMP increase induced by adenosine analogues and that this is due to differences in the amount of adenosine A2A receptor, G protein and effector. A large difference in receptor number resulted in differences in potency of an agonist.
Insights
PC12 cell clones exhibit significant variations in adenosine A2A receptor signaling. These differences stem from distinct levels of A2A receptors, G proteins, and effectors, impacting cellular responses to adenosine analogues.
Area of Science:
- Neuroscience
- Molecular Biology
- Pharmacology
Background:
- PC12 cells are a widely used neuronal model system.
- Wild-type PC12 cells are genetically heterogeneous, existing as multiple subclones.
- Understanding receptor signaling in these subclones is crucial for reproducible research.
Purpose of the Study:
- To investigate the signal transduction pathways of the A2A adenosine receptor in different PC12 cell subclones.
- To characterize variations in receptor expression, G protein coupling, and functional responses among PC12 clones.
- To elucidate the molecular basis for differential cellular responses to adenosine receptor agonists.
Main Methods:
- Reverse transcription polymerase chain reaction (RT-PCR) to detect adenosine receptor mRNA.
- Radioligand binding assays using [3H]SCH 58261 to quantify A2A receptor density (Bmax).
- Measurement of G protein (Gi and Gs) levels and cAMP production in response to adenosine receptor agonists (NECA, CGS 21680).
Main Results:
- All four PC12 clones (1, 19, 21, 27) expressed A2A, A2B, and A1 adenosine receptor mRNAs, but not A3.
- A2A receptor density (Bmax) varied significantly, being highest in clone 1 and lowest in clone 27.
- Functional responses to adenosine analogues correlated with A2A receptor levels, with lower potency (higher EC50) observed in clone 27.
- Differences in Gs protein levels were noted between clones, potentially contributing to signal transduction variations.
Conclusions:
- PC12 cell subclones display substantial heterogeneity in A2A adenosine receptor expression and function.
- Variations in A2A receptor number, G protein composition, and effector systems underlie differential cAMP responses.
- Receptor density is a key determinant of agonist potency in these PC12 cell models.