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 ra te of cultured astrocytes by measuring H-3-thymidine incorporation and by flow cytometric analysis of the cell cycle. Determinations after 1 6 hours showed that ATP present in the culture medium for the whole pe riod caused a dose-dependent reduction of cell proliferation, while if the exposure to ATP was limited to the first 8 hours, the proliferati on was increased (always in a dose-dependent manner). A time course st udy 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 t he 8th hour, was followed by a H-3-thymidine incorporation occurring a t such a fast rate to overshoot the control values. High performance l iquid chromatography (HPLC) analysis, carried out to identify purine c ompounds present in the culture medium during cell exposure to ATP, in dicated that more than 95% of the added ATP was metabolized within 1 h r. Conversely, an increase of purine metabolites was measured, this ac cumulation being greater at the highest concentrations of added ATP. T he 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 confir med 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 me tabolized as fast as ATP, reproduced effects very similar to the ATP-i nduced ones. On the other hand, the nonhydrolisable ATP analogue, aden osine 5'-(beta, gamma-imido)-triphosphate (AMP-PNP) at 100 mu M, induc ed a mitogenic effect as well as the A(2) site stimulation. On the con trary, the activation of A(1) receptors by 5 mu M R-phenyl-isopropylad enosine (R-PIA) inhibited astrocyte proliferation; moreover, 100 nM 8- cyclopentyl-1,3-dipropylxanthine (DPCPX), an A(1) site antagonist, rev ersed the ATP-induced inhibition of cell proliferation. These results indicate that exogenous ATP, as a consequence of its rapid extracellul ar breakdown, exerts a dual influence on astrocyte proliferation by th e 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 pu rines released in the brain can influence a reactive astrocyte prolife rative response to injury. (C) 1994 Wiley-Liss, Inc.