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

Abstract

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 0.45µm 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. 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. 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. 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 ƒF (n = 356) in controls to 7.9 ± 0.2 ƒF 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. The results suggest that a cAMP-dependent mechanism mediates the fusion of larger granules with the plasmalemma.