MODULATION OF THE UNITARY EXOCYTIC EVENT AMPLITUDE BY CAMP IN RAT MELANOTROPHS

1. Secretory responses were measured in single rat pituitary melanotro phs 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 th e added presence of cAMP (0.2 mM) in the patch pipette solution, capac itance 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 t o intact melanotrophs and [Ca2+](i) was measured using fura-2 AM. Addi tion 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 activi ty 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 secretor y granule with the plasmalemma was monitored by measuring discrete fem tofarad steps in C-m. The signal-to-noise ratio of recordings was incr eased by preincubating the cells with a hydrophobic anion, dipicrylami ne. 4. Recordings of unitary exocytic events (discrete 'on' steps in C -m) showed that the amplitude of 'on' steps - a parameter correlated t o 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 wa s similar in controls and in the presence of cAMP. 5. The results sugg est that a cAMP-dependent mechanism mediates the fusion of larger gran ules with the plasmalemma.