Dual Effect of Adenosine on Vascular Smooth Muscle [³H]-Thymidine DNA Labeling: Receptor-Mediated Modulation of DNA Synthesis and Inhibition of Thymidine Uptake

Abstract

This study examined the contribution of cAMP signaling to the modulation of vascular smooth muscle cell (VSMC) proliferation by adenosine. At a concentration of 1 mM, adenosine inhibited [³H]-thymidine uptake, measured as the initial rate of isotope influx, by 10-fold. Diminution of [³H]-thymidine uptake by adenosine was independent of the presence of A₁- and A₂-receptor antagonists, indicating that adenosine competes with thymidine for plasma membrane transporter-binding sites. Considering these results, in order to estimate [³H]-thymidine DNA labeling, VSMCs were preincubated with adenosine for 48 h, and adenosine was then omitted during the subsequent 2 h of incubation in [³H]-thymidine-containing medium. In serum-depleted VSMCs, preincubation with 100 µM or 1,000 µM adenosine augmented DNA synthesis by approximately 6- and 3-fold, respectively, whereas the increment of DNA synthesis triggered by serum was decreased in the presence of adenosine by 20–30%. Both cAMP production and inhibition of DNA synthesis by adenosine in serum-supplied cells were independent of the presence of the A₁-antagonist 1,2-dipropyl-8-cyclopentylxanthine (DPCPX), but were abolished by the A₂-antagonist 1,3-dimethyl-7-propylxanthine (DMPX). In contrast, the activation of DNA synthesis in serum-depleted cells by adenosine was decreased in the presence of DPCPX and DMPX by approximately 30 and 40%, respectively. Both in serum-supplied and -depleted VSMCs, dose-dependent elevation of cAMP production with an adenylate cyclase activator, forskolin, reduced DNA synthesis by up to 40–60%. Thus, our results show that in addition to suppressing thymidine uptake, adenosine depresses the DNA synthesis triggered by serum-derived growth factors and stimulates DNA synthesis in serum-depleted cells. These data also suggest that the inhibition of DNA synthesis is mediated by cAMP production where the activation of DNA synthesis is independent of cAMP signaling.

A Novel Role for Raloxifene Nanomicelles in Management of Castrate Resistant Prostate Cancer

“…Briefly, PC3 (20,000 cells/well) and DU145 cells (8 × 10 4  cells/well) were seeded in 24 well-plates and incubated for 36 h, cells were treated with SMA-Ral or free raloxifene at 2, 5, and 10  μ M for 48 h. [ 3 H]-thymidine (0.5  μ Ci/well) was added for the last 20 h of the treatment. [ 3 H]-thymidine incorporation was measured as described previously [32]. …”

ERK, PKC and PI3K/Akt Pathways Mediate Extracellular ATP and Adenosine-Induced Proliferation of U138-MG Human Glioma Cell Line

“…Fixed cells were kept at 4°C until the assay was performed. At time of use, cells were washed once with PBS and incubated for 30 min in the dark with 1 ml of ice-cold buffer containing 3.4 m M sodium citrate, 0.1 mg/ml Rnase A and 0.02 mg/ml propidium iodide, after which the cells were assayed in a Beckman Dickinson FACS cytometer using Cell Quest analysis [24]. …”