Abstract
Cyclic AMP analogs containing hydrophobic modification of C(8) at the adenine ring such as 8-(4-chlorophenylthio)-cAMP (8-pCPT-cAMP) and 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-methyl-cAMP) can penetrate membranes due to their high lipophilicity and directly activate intracellular cAMP effectors. Therefore, these cAMP analogs have been used in numerous studies, assuming that their effects reflect the consequences of direct activation of cAMP effectors. The present study provides evidence that 8-pCPT-modified cAMP analogs and their corresponding putative hydrolysis products (8-(4-chlorophenylthio)-adenosine (8-pCPT-ado) and 8-(4-chlorophenylthio)-2'-O-methyl-adenosine (8-pCPT-2'-O-methyl-ado)) inhibit the equilibrative nucleoside transporter 1 (ENT1). In PC12 cells, in which nucleoside transport strongly depended on ENT1, 8-pCPT-ado, 8-pCPT-2'-O-methyl-ado, and, to a smaller extent, 8-pCPT-2'-O-methyl-cAMP caused an increase of protein kinase A substrate motif phosphorylation and anti-apoptotic effect by an A(2A) adenosine receptor (A(2A)R)-dependent mechanism. In contrast, the effects of 8-pCPT-cAMP were mainly A(2A)R-independent. In HEK 293 showing little endogenous ENT1-dependent nucleoside transport, transfection of ENT1 conferred A(2A)R-dependent increase in protein kinase A substrate motif phosphorylation. Together, the data of the present study indicate that inhibition of ENT1 and activation of adenosine receptors have to be considered when interpreting the effects of 8-pCPT-substituted cAMP/adenosine analogs.
Highlights
Given the important role of cAMP in cell proliferation [1], we initially studied the effect of membrane-permeable Epac-selective and non-selective cAMP analogs on cell proliferation
We show that 8-permeable cAMP analogs containing the hydrophobic 4-chlorophenylthio (pCPT)-modified cAMP analogs, and more potently, 8pCPT-modified adenosine analogs, cause inhibition of the equilibrative nucleoside transporter 1 (ENT1) and activation of Gs protein-coupled A2A adenosine receptors (A2AR) by an ENT1-dependent mechanism
8-pCPT-modified cAMP/Ado Analogs Inhibit ENT1—Because of the discrepancy of the effect of 8-pCPT-modified cAMP analogs on [3H]thymidine incorporation and cell number, we examined whether the inhibitory effect of C8-modified cAMP/ado analogs on [3H]thymidine incorporation could be due to inhibition of [3H]thymidine transport into the cells. 8-pCPT-cAMP, 8-pCPT-2Ј-O-methyl-cAMP, as well as their Sp and adenosine analogs strongly inhibited [3H]thymidine uptake into PC12 cells (Fig. 3, A–C), 8-pCPT-ado having the strongest effect
Summary
Reagents—Adenosine, cAMP, SCH 58261, 5-(4-nitrobenzyl)6-thioinosine (NBMPR), and anti-actin IgG were obtained from Sigma-Aldrich. HEK 293 and Panc-1 cells were cultured in DMEM supplemented with 10% fetal calf serum and penicillin/ streptomycin. Primary human mesangial cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum and antibiotics [12]. [3H]Thymidine Incorporation into DNA—Cells grown on 96-well plates were incubated with the cAMP/adenosine analogs for the indicated time. [3H]Thymidine Uptake—Cells were grown on 24-well plates and were switched to serum-free DMEM 4 h prior to the experiment. After preincubation of the cells with the nucleotide/nucleoside analogs for 30 min, 0.3 Ci of [3H]thymidine was added, and the incubation was continued for 1.5 min (PC12 and renal mesangial cells) or 15 min (HEK 293 cells). Apoptosis Assay—Cells grown in 24-well dishes were switched to serum-free DMEM, and 8-pCPT-cAMP/ado and/or ADA were added to the medium. Data from different treatment groups were analyzed by the Student’s t test for unpaired samples. p values smaller than 0.05 were considered to indicate statistical significance (*) (see Figs. 1– 6)
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