Protein kinase A‐dependent and ‐independent stimulation of exocytosis by cAMP in mouse pancreatic B‐cells

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Abstract
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 (≥60 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.