Abstract：
:Cyclic AMP regulates the late step of Ca²+-dependent exocytosis in many secretory cells through two major mechanisms: a protein kinase A-dependent and a cAMP-GEF/Epac-dependent pathway. We designed a protocol to characterize the role of these two cAMP-dependent pathways on the Ca²+ sensitivity and kinetics of regulated exocytosis in mouse pancreatic beta cells, using a whole-cell patch-clamp based capacitance measurements. A train of depolarizing pulses or slow photo-release of caged Ca²+ were stimuli for the exocytotic activity. In controls, due to exocytosis after slow photo-release, the C(m) change had typically two phases. We observed that the Ca²+-dependency of the rate of the first C(m) change follows saturation kinetics with high cooperativity and half-maximal rate at 2.9±0.2 μM. The intracellular depletion of cAMP did not change amp1, while rate1 and amp2 were strongly reduced. This manipulation pushed the Ca²+-dependency of the exocytotic burst to significantly lower [Ca²+](i). To address the question of which of the cAMP-dependent mechanisms regulates the observed shifts in Ca²+ dependency we included regulators of PKA and Epac2 activity in the pipette solution. PKA activation with 100 μM 6-Phe-cAMP or inhibition with 500 μM Rp-cAMPs in beta cells significantly shifted the EC(50) in the opposite directions. Specific activation of Epac2 did not change Ca²+ sensitivity. Our findings suggest that cAMP modulates Ca²+-dependent exocytosis in mouse beta cells mainly through a PKA-dependent mechanism by sensitizing the insulin releasing machinery to [Ca²+](i); Epac2 may contribute to enhance the rates of secretory vesicle fusion.
journal_name
Cell Calciumjournal_title
Cell calciumauthors
Skelin M,Rupnik Mdoi
10.1016/j.ceca.2010.12.005subject
Has Abstractpub_date
2011-02-01 00:00:00pages
89-99issue
2eissn
0143-4160issn
1532-1991pii
S0143-4160(10)00206-Xjournal_volume
49pub_type
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