Adenosine A2A and ATP receptors in PC12 cells

Sammanfattning: PC12 cells, originally isolated from a rat pheochromocytoma, are widely used to study receptor pharmacology and nerve cell differentiation. They are the neoplastic counterpart of adrenal chromaffin cells, but upon treatment with NGF they acquire a neuronal phenotype. The aim of this thesis was to characterize adenosine and ATP receptor expression in undifferentiated and NGF-differentiated PC12 cells. ATP, the major intracellular high energy compound, is as important intracellularly as extracellularly, where it acts as a signal molecule. ATP can be broken down to adenosine, which is a neurohumoral agent and an autacoid. It is well known that PC12 cells can undergo spontaneous mutations. To identify a reliable clone, the expression of adenosine receptors was examined in four PC12 cell clones, previously isolated from a common parental cell line. Functional responses to adenosine analogues were attributable to A2A receptors in all four clones. The magnitude of responses appeared to be correlated to the abundance of adenylyl cyclase, whereas the potency of the agonists was determined by the amount of receptor protein. One of the clones was chosen for further studies. NGF differentiation of these cells led to down-regulation of A2A receptors, probably via reduction of the transcription of the receptor gene. Despite the presence of mRNAs for several P2 receptors, responses to ATP in undifferentiated PC 12 cells were attributable to P2X4 receptors only. There was functional evidence for P2X2, P2X4 and P2Y2 receptors in NGF-differentiated cells. The role of adenosine receptors in nerve cells during cell stress was examined by exposing PC12 cells to hypoxia. Adenosine and neurotrophins can be released in conditions of cell stress, and MAP kinase phosphorylation was employed as a marker to study interactions between their signal transduction pathways. A2A receptor activation exerted a biphasic effect on NGF-induced MAP kinase activation: an initial inhibition was followed by an additive effect. The two effects are probably mediated by the two kinases Raf1 and B-Raf, respectively. When undifferentiated PC12 cells were subjected to hypoxia, endogenous adenosine conveyed a protective effect, as an A2A antagonist decreased the viability of the cells. This effect was not seen in NGF-treated cells. Hypoxia also increased the expression of A2A receptors in undifferentiated cells via a process of redistribution from the cytoplasm to the cell membrane. In conclusion, PC12 cells proved to be a useful model for studies of adenosine and ATP receptors in vitro. NGF-differentiation induces profound changes, in opposite directions, in the expression of adenosine A2A and ATP receptors, where the former are reduced while the latter are increased by expression of another type of functional receptor. This may be functionally important: whereas P2X receptors provide only a short lasting signal in undifferentiated cells, activation of P2Y receptors in NGF treated PC12 cells results in long lasting effects. In addition, NGF can down-regulate A2A receptors, and these receptors can, in turn, inhibit NGF signaling. Finally, we found that hypoxia induces an increase in A2A receptors via a novel mechanism, and confirmed that, even in this model, adenosine exerts a protective effect.