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Fig. 5. Congruence between time dependency of P2Y₁R stimulation for cell proliferation and half-life of injury-released nucleotides. (A) Stimulation time-period necessary for P2Y₁R-induced cell proliferation. FRT cells were incubated with 1 µM 2-MeSADP for the indicated time periods and then the ligand was removed and the cells incubated for 16 hours with [³H]thymidine. The proliferative response requires a minimal stimulation time of 10 minutes, reaching a maximum by 120 minutes and decreases thereafter (n=4); (B) Changes in cyclin D levels detected by immunoblot. FRT cells were incubated either with carrier culture medium alone (Control, C) or with 1 µM 2-MeSADP for the indicated times. Stimulation for as little as 15 minutes leads to increased levels of cyclin D, analyzed 16 hours later. The quantification of three separate assays normalized with respect to actin is shown in the graph. (C) Half-life of nucleotides released from wounded cultured cells. Extracellular ATP and ADP concentrations show a dramatic increase after cell injury and then gradually decrease. ADP maintained higher levels for longer periods – enough to induce P2Y₁R-mediated cell proliferation (n=4). Values are the means ± s.e.m. ^*P<0.05, ^**P<0.01; Dunnett's test against control conditions without agonist.

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