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 μ m Ca 2+ . 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 ([Ca 2+ ] i ), dibutyryl cyclic AMP (dbcAMP, 10 mM) was added to intact melanotrophs and [Ca 2+ ] i was measured using fura‐2 AM. Addition of dbcAMP caused a transient reduction in [Ca 2+ ] 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 [Ca 2+ ] 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 pre‐incubating 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.