Fig. 9. MDCK cells ectopically expressing the P2Y₁R display lower doubling times and EGFR-dependent increased proliferation rates. (A) Viability curves for wild-type (wt) MDCK cells, or cells stably transfected with either the P2Y₁R (MDCK/P2Y₁R) or -opioid receptor (MDCK/-opioid). The number of cells increases with higher slope and reaches a higher maximum for MDCK/P2Y1R cells, whereas cells expressing ectopic -opioid receptor behave like wild-type MDCK cells. MDCK P2Y₁R cells also show a reduced doubling time. Values are the means ± s.e.m. (n=4-6). (B) Basal proliferation rate of MDCK P2Y₁R is higher than wild-type cells and sensitive to 0.1 µM AG1478, thus indicating a dependency on EGFR activity. In MDCK P2Y₁R cells, inhibition of EGFR activity significantly decreased the [³H]thymidine incorporation. Cells were treated with 100 nM AG1478 during 16 hours of [³H]thymidine incorporation. Values are the means ± s.e.m. ^*P<0.05, ^**P<0.01; Dunnett's test compared with control wild-type cells (n=6). (C) The selective P2Y₁R agonist 2-MeSADP increases proliferation of MDCK/P2Y₁R cells in a concentration-dependent manner. Cells were treated with exogenous 2-MeSADP for 1 hour before [³H]thymidine incorporation. Values are the means ± s.e.m. ^**P<0.01; Dunnett's test compared with untreated cells (n=3). (D) P2Y₁R transactivates the EGFR in MDCK P2Y₁R cells. The immunoblot shows an increased EGFR tyrosine phosphorylation in cells treated with either 10 nM 2-MeSADP or 1 nM EGF for 5 minutes, quantified with respect to total EGFR mass and depicted below each lane. Only the carrier culture medium was added for controls.