Membrane-permeant derivatives of cyclic AMP optimized for high potency, prolonged activity, or rapid reversibility.
A novel membrane-permeant derivative of cAMP, cAMP acetoxymethyl ester (cAMP/AM), was synthesized via silylated intermediates. Its ability to induce Cl- secretion by T84 cells, a human colon cancer cell line, was compared with that of two other membrane-permeant cAMP derivatives that were recently introduced, N6,O2'-dibutyryl-cAMP acetoxymethyl ester (bt2cAMP/AM) and Sp-5,6-dichlorobenzimidazole-1-beta-D-ribofuranoside 3',5'-cyclic phosphorothioate (Sp-5,6-DCl-cBIMPS). All of these compounds are powerful activators of Cl- secretion when applied extracellularly, with EC50 values of 60 microM, 0.7 microM, and 3 microM, respectively. However, cAMP/AM was expected to be readily degraded inside cells, in contrast to the cyclophosphodiesterase-resistant Sp-5,6-DCI-cBIMPS or the only slowly metabolizable N6-butyryl-cAMP derived from bt2cAMP/AM. Reversibility of cAMP/AM action was demonstrated by wash-out experiments; Cl- secretion induced by high doses of cAMP/AM (100 microM) could be quickly abolished by rinsing of the cells, whereas similar experiments with bt2cAMP/AM and Sp-5,6-DCI-cBIMPS showed much slower decreases. Even more sensitive to residual cAMP derivatives was the synergistic effect of carbachol, which was applied after the incubation with membrane-permeant derivatives and their subsequent wash-out. Although doses of cAMP derivatives that barely activated Cl- secretion were readily capable of inducing a synergistic response with carbachol, cells incubated with high doses of cAMP/AM (100 microM) and subsequently washed showed only a nonsynergistic carbachol response, in contrast to cells incubated with bt2cAMP/AM or Sp-5,6-DCI-cBIMPS. We therefore characterize cAMP/AM as a membrane-permeant derivative of cAMP that is easily metabolizable inside cells and hence is most useful for applications where a transient intracellular cAMP signal is desired. In contrast, completely nonmetabolizable Sp-5,6-DCI-cBIMPS seems to be more useful in longer incubations that require steady levels of cAMP-dependent protein kinase activation. bt2cAMP/AM combines the advantages of intracellular trapping by ester hydrolysis and reduced cyclophosphodiesterase sensitivity of its active intracellular product, which probably lead to its particularly high potency.
Schultz
C
C
Department of Pharmacology, University of California, San Diego, La Jolla 92093.
Vajanaphanich
M
M
Genieser
H G
HG
Jastorff
B
B
Barrett
K E
KE
Tsien
R Y
RY
D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002712:Chlorides / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D014407:Tumor Cells, Cultured
D002712:Chlorides; D000242:Cyclic AMP