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

Abstract

1. Secretory responses were measured in single rat pituitary melanotrophs as the relative increase in membrane capacitance (Cm) 8 min after the start of dialysis with solutions containing 0.45 microM 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 Cm. 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 Cm) 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.