Figure 2. Effects of caffeine on cAMP secretion and cAMP PDE activity.

(A) cAMP secretion. Vegetative aca–/rdeA– cells were incubated at 10⁸ cells/ml with 5 mM DTT in PB in the presence and absence of 2 mM caffeine. At the indicated time periods 50 μl aliquots of cell suspension were centrifuged for 5 s at 10000 g. Supernatant (medium) and pellet (cells) fractions were rapidly separated and boiled for 30 s. cAMP was assayed in both fractions. (B) cAMP production in PDE-null mutants. Vegetative pdeE–/regA– cells were incubated for 30 min with 10 mM DTT in PB in the presence and absence of 3 mM IBMX and the indicated caffeine concentrations and assayed for cAMP. Data are expressed as percentage of cAMP accumulation in the absence of IBMX and caffeine. (C) PDE activity. Vegetative wild-type cells were incubated with 10 mM DTT and 10⁻⁷ M [³H]cAMP in the presence and absence of IBMX and caffeine as indicated. After 30 min the samples were assayed for [³H]5′-AMP production. Data are presented as percentage of [³H]cAMP hydrolysis obtained in the absence of IBMX and caffeine. Means±S.E.M. for two experiments performed in duplicate for (A) and triplicate for (B, C) are presented.