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 t o induce Cl- secretion by T84 cells, a human colon cancer cell line, w as compared with that of two other membrane-permeant cAMP derivatives that were recently introduced, N-6,O-2'-dibutyryl-cAMP acetoxymethyl e ster (bt(2)cAMP/AM) and -5,6-dichlorobenzimidazole-1-beta-D-ribofurano side 3',5'-cyclic phosphorothioate (S-p-5,6-DCl-cBIMPS). All of these compounds are powerful activators of Cl- secretion when applied extrac ellularly, with EC(50) values of 60 mu M, 0.7 mu M, and 3 mu M, respec tively. However, cAMP/AM was expected to be readily degraded inside ce lls, in contrast to the cyclophosphodiesterase-resistant S-p-5,6-DCl-c BIMPS or the only slowly metabolizable N-6-butyryl-cAMP derived from b t(2)cAMP/AM. Reversibility of cAMP/AM action was demonstrated by wash- out experiments; Cl- secretion induced by high doses of cAMP/AM (100 m u M) could be quickly abolished by rinsing of the cells, whereas simil ar experiments with bt(2)cAMP/AM and S-p-5,6-DCl-cBIMPS showed much sl ower decreases. Even more sensitive to residual cAMP derivatives was t he synergistic effect of carbachol, which was applied after the incuba tion with membrane-permeant derivatives and their subsequent wash-out. Although doses of cAMP derivatives that barely activated Cl- secretio n were readily capable of inducing a synergistic response with carbach ol, cells incubated with high doses of cAMP/AM (100 mu M) and subseque ntly washed showed only a nonsynergistic carbachol response, in contra st to cells incubated with bt(2)cAMP/AM or S-p-5,6-DCl-cBIMPS. We ther efore characterize cAMP/AM as a membrane-permeant derivative of cAMP t hat is easily metabolizable inside cells and hence is most useful for applications where a transient intracellular cAMP signal is desired. I n contrast, completely nonmetabolizable S-p-5,6-DCl-cBIMPS seems to be more useful in longer incubations that require steady levels of cAMP- dependent protein kinase activation. bt(2)cAMP/AM combines the advanta ges of intracellular trapping by ester hydrolysis and reduced cyclopho sphodiesterase sensitivity of its active intracellular product, which probably lead to its particularly high potency.