• 1
    The mechanisms by which cAMP stimulates Ca2+-dependent insulin secretion were investigated by combining measurements of whole-cell Ca2+ currents, the cytoplasmic free Ca2+ concentration ([Ca2+]i) and membrane capacitance in single mouse B-cells maintained in tissue culture.
  • 2
    Cyclic AMP stimulated exocytosis > 4-fold in whole-cell experiments in which secretion was evoked by intracellular dialysis with a Ca2+-EGTA buffer with a [Ca2+]i of 1.5μm. This effect was antagonized by inhibitors of protein kinase A (PKA).
  • 3
    Photorelease of cAMP from a caged precursor potentiated exocytosis at Ca2+ concentrations which were themselves stimulatory (geqslant R: gt-or-equal, slanted60 nm), but was without effect in the complete absence of Ca2+.
  • 4
    Elevation of intracellular cAMP (by exposure to forskolin) evoked a 6-fold PKA-dependent enhancement of the maximal exocytotic response (determined as the maximum increase in cell capacitance that could be elicited by a train of depolarizations) in perforated-patch whole-cell recordings.
  • 5
    Exocytosis triggered by single depolarizations in standard whole-cell recordings was strongly potentiated by cAMP, but in this case the effect was unaffected by PKA inhibition.
  • 6
    When exocytosis was triggered by Ca2+ released from Ca2+-NP-EGTA (‘caged Ca2+), cAMP exerted a dual stimulatory effect on secretion: a rapid (initiated within 80 ms) PKA-independent phase and a late PKA-dependent component.
  • 7
    We conclude that cAMP stimulates insulin secretion both by increasing the release probability of secretory granules already in the readily releasable pool and by accelerating the refilling of this pool.