cAMP increases the sensitivity of exocytosis to Ca²+ primarily through protein kinase A in mouse pancreatic beta cells.
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.
Faculty of Medicine University of Maribor, Slomskov trg 15, 2000 Maribor, Slovenia.
Maša
M
Skelin
Marjan
M
Rupnik
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D025802:Electric Capacitance; D005089:Exocytosis; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000201:enzymology / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D018408:Patch-Clamp Techniques
D002352:Carrier Proteins; D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin; C486707:Rapgef4 protein, mouse; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium