Inhibition of the equilibrative nucleoside transporter 1 and activation of A2A adenosine receptors by 8-(4-chlorophenylthio)-modified cAMP analogs and their hydrolytic products.
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.
Department of Medicine I, University Hospital Frankfurt, D-60590 Frankfurt, Germany.
Oliver
O
Waidmann
Thomas
T
Pleli
Karel
K
Dvorak
Christina
C
Baehr
Ulrich
U
Mondorf
Guido
G
Plotz
Ricardo M
RM
Biondi
Stefan
S
Zeuzem
Albrecht
A
Piiper
D020816:Amino Acid Motifs; D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D004396:Coloring Agents / Q000494:pharmacology; D000242:Cyclic AMP / Q000737:chemistry* / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry; D033721:Equilibrative Nucleoside Transporter 1 / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D016716:PC12 Cells; D010766:Phosphorylation; D051381:Rats; D043705:Receptor, Adenosine A2A / Q000737:chemistry*; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology
D004396:Coloring Agents; D033721:Equilibrative Nucleoside Transporter 1; D043705:Receptor, Adenosine A2A; D013778:Tetrazolium Salts; D013844:Thiazoles; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; C022616:thiazolyl blue