Modulation of the unitary exocytic event amplitude by cAMP in rat melanotrophs

1. Secretory responses were measured in single rat pituitary melanotrophs as the relative increase in membrane capacitance (C-m) 8 min after the start of dialysis with solutions containing 0.45 mu M Ca2+. In the added presence of cAMP (0.2 mM) in the patch pipette solution, capacitance responses increased 2- to 3-fold in comparison with controls. 2. To study whether cAMP-dependent mechanisms affect cytosolic calcium activity ([Ca2+](i)), dibutyryl cyclic AMP (dbcAMP, 10 mM) was added to intact melanotrophs and [Ca2+](i) was measured using fura-2 AM. Addition of dbcAMP caused a transient reduction in [Ca2+](i) to 82 +/- 21 nM from a resting value of 100 +/- 19 nM (mean +/- S.E.M., n = 32, P < 0.002), indicating that the cAMP-induced increase in secretory activity was not the result of cAMP acting to increase [Ca2+](i), which then increased secretory activity. 3. To investigate whether cAMP affects the secretory apparatus directly, the interaction of a single secretory granule with the plasmalemma was monitored by measuring discrete femtofarad steps in C-m. The signal-to-noise ratio of recordings was increased by preincubating the cells with a hydrophobic anion, dipicrylamine. 4. Recordings of unitary exocytic events (discrete 'on' steps in C-m) showed that the amplitude of 'on' steps - a parameter correlated to the size of exocytosing secretory granules increased from 4.2 +/- 0.2 fF (n = 356) in controls to 7.9 +/- 0.2 fF in the presence of cAMP (n = 329, P < 0.001), while the frequency of unitary exocytic events was similar in controls and in the presence of cAMP. 5. The results suggest that a cAMP-dependent mechanism mediates the fusion of larger granules with the plasmalemma.