Membrane-permeant derivatives of cyclic AMP optimized for high potency, prolonged activity, or rapid reversibility

Carsten Schultz, Mana Vajanaphanich, Hans Gottfried Genieser, Bernd Jastorff, Kim E. Barrett, Roger Y. Tsien

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Abstract

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 S(p)-5,6-dichlorobenzimidazole-1-β-D-ribofuranoside 3',5'-cyclic phosphorothioate (S(p)-5,6-DCl-cBIMPS). All of these compounds are powerful activators of Cl- secretion when applied extracellularly, with EC50 values of 60 μM, 0.7 μM, and 3 μM, respectively. However, cAMP/AM was expected to be readily degraded inside cells, in contrast to the cyclophosphodiesterase- resistant S(p)-5,6-DCl-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 μM) could be quickly abolished by rinsing of the cells, whereas similar experiments with bt2cAMP/AM and S(p)-5,6-DCl-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 μM) and subsequently washed showed only a nonsynergistic carbachol response, in contrast to cells incubated with bt2cAMP/AM or S(p)-5,6-DCl-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 S(p)-5,6-DCl-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.

Original languageEnglish (US)
Pages (from-to)702-708
Number of pages7
JournalMolecular Pharmacology
Volume46
Issue number4
StatePublished - Oct 1994
Externally publishedYes

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ASJC Scopus subject areas

  • Pharmacology

Cite this

Schultz, C., Vajanaphanich, M., Genieser, H. G., Jastorff, B., Barrett, K. E., & Tsien, R. Y. (1994). Membrane-permeant derivatives of cyclic AMP optimized for high potency, prolonged activity, or rapid reversibility. Molecular Pharmacology, 46(4), 702-708.