JBC, Vol. 251, Issue 21, 6757-6766, Nov, 1976
2-Fluoroadenosine 3':5'-monophosphate. A metabolite of 2-fluoroadenosine in mouse cytotoxic lymphocytes
T. P. Zimmerman, J. L. Rideout, G. Wolberg, G. S. Duncan and G. B. Elion
2-Fluoroadenosine (F-Ado) is a potent inhibitor of lymphocyte-mediated
cytolysis studied in vitro. The inhibition of cytolysis by F-Ado was
potentiated markedly by an inhibiotr (Ro 20-1724) of adenosine
3':5'-monophosphate (cAMP) phosphodiesterase and, unlike the inhibition
caused by adenosine, was irreversible when the cytotoxic lymphocytes were
incubated with F-Ado and were then washed free of exogenous nucleoside.
Incubation of cytotoxic lymphocytes with F-Ado resulted in the rapid,
dose-dependent formation of 2-fluoroadenosine 5'-triphosphate (F-ATP); the
build-up of F-ATP within these cells was accompanied by a reciprocal
depletion of ATP. Once formed intracellularly, the F-ATP was not diminished
during a subsequent 30-min incubation of the cells in F-Ado-free medium.
2-Fluoroadenosine 3':5'-monophosphate (F-cAMP), a novel compound, was
synthesized chemically. This cAMP analogue was found to be highly
cross-reactive in a radioimmunoassay specific for cAMP and to be equipotent
to cAMP in its ability to activate a crude preparation of protein kinase
derived from rat brain. A column chromatographic procedure was devised
whereby F-cAMP and cAMP could be purified simultaneously from tissue
extracts. Treatment of cytotoxic lymphocytes with F-Ado resulted in the
formation of presumptive F-cAMP in amounts greater than that of cAMP, as
determined by the concentration of F-Ado added to the medium and was not
observed when the lymphocytes were incubated with either adenosine or
2-chloroadenosine, two agents which caused large increases in cAMP. The
simultaneous presence of Ro 20-1724 enhances greatly the formation of
F-cAMP from F-Ado without affecting the pool size of F-ATP. Removal of
exogenous F-Ado from cells previously incubated with this drug and
subsequent incubation of these cells in drug-free medium did not result in
a substantial reduction in intracellular F-Ado (via prior incubation with
F-Ado); 2'-deoxyadenosine was also effective in this capacity, while
9-beta-D-arabinofulanosyladenine was without effect. The level of cAMP was
elevated transiently, in a dose-dependent manner, by F-Ado, and returned to
control value after removal of exogenous F-Ado from the cells. Ro 20-1724
enhanced greatly this transient elevation of cAMP caused by F-Ado.
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