EFFECTS OF EXOGENOUS ATP AND RELATED ANALOGS ON THE PROLIFERATION RATE OF DISSOCIATED PRIMARY CULTURES OF RAT ASTROCYTES

The effects of ATP (5-500 mu M) were evaluated on the proliferation rate of cultured astrocytes by measuring H-3-thymidine incorporation and by flow cytometric analysis of the cell cycle. Determinations after 16 hours showed that ATP present in the culture medium for the whole period caused a dose-dependent reduction of cell proliferation, while if the exposure to ATP was limited to the first 8 hours, the proliferation was increased (always in a dose-dependent manner). A time course study of 3H-thymidine incorporation showed that, in the presence of ATP, H-3-thymidine was incorporated at a slower rate than in controls; the replacement of the culture medium with an ATP-free fresh medium, at the 8th hour, was followed by a H-3-thymidine incorporation occurring at such a fast rate to overshoot the control values. High performance liquid chromatography (HPLC) analysis, carried out to identify purine compounds present in the culture medium during cell exposure to ATP, indicated that more than 95% of the added ATP was metabolized within 1 hr. Conversely, an increase of purine metabolites was measured, this accumulation being greater at the highest concentrations of added ATP. The presence of high levels of extracellular ATP catabolites suggested that these compounds may act on the regulation of cell replication via the different purine receptors. This hypothesis was tested and confirmed by using agonists and antagonists selective for the P-1 and the P-2 sites. One hundred mu M 2methylthio-ATP (2MeSATP), a P-2Y agonist metabolized as fast as ATP, reproduced effects very similar to the ATP-induced ones. On the other hand, the nonhydrolisable ATP analogue, adenosine 5'-(beta, gamma-imido)-triphosphate (AMP-PNP) at 100 mu M, induced a mitogenic effect as well as the A(2) site stimulation. On the contrary, the activation of A(1) receptors by 5 mu M R-phenyl-isopropyladenosine (R-PIA) inhibited astrocyte proliferation; moreover, 100 nM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an A(1) site antagonist, reversed the ATP-induced inhibition of cell proliferation. These results indicate that exogenous ATP, as a consequence of its rapid extracellular breakdown, exerts a dual influence on astrocyte proliferation by the involvement of both P-1 and P-2Y receptors. These findings might be relevant to such pathological conditions of the central nervous system (CNS), as seizures, hypoxia or ischemia, in which great amounts of purines released in the brain can influence a reactive astrocyte proliferative response to injury. (C) 1994 Wiley-Liss, Inc.