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 (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.
Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560012, India.
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Sikdar
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Kreft
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Zorec
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology*; D003594:Cytoplasmic Granules / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004553:Electric Conductivity; D004594:Electrophysiology; D005089:Exocytosis / Q000502:physiology*; D008297:Male; D008544:Melanocytes / Q000166:cytology* / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D010851:Picrates / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
D010851:Picrates; C011454:dipicrylamine; D003994:Bucladesine; D000255:Adenosine Triphosphate; D000242:Cyclic AMP