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cAMP increases the sensitivity of exocytosis to Ca2+ primarily through protein kinase A in mouse pancreatic beta cellsSkelin, Maša ; Rupnik, MarjanCyclic AMP regulates the late step of Ca2+-dependent exocytosis in many secretory cells through two major mechanisms: a protein kinase A-dependent a nda 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 Cm change had typically two phases. We observed that the Ca2+-dependency of the rate of the first Cm change follows saturation kinetics with high cooperativity and half-maximal rate at 2.9 0.2 M. 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 M 6-Phe-cAMP or inhibition with 500 M Rp-cAMPs in beta cells significantly shifted the EC50 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.Vir: Cell calcium. - ISSN 0143-4160 (Vol. 49, issue 2, 2011, str. 89-99)Vrsta gradiva - članek, sestavni delLeto - 2011Jezik - angleškiCOBISS.SI-ID - 66514689
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Ime baze podatkov | Področje | Leto |
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Povezave do osebnih bibliografij avtorjev | Povezave do podatkov o raziskovalcih v sistemu SICRIS |
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Skelin, Maša | 29565 |
Rupnik, Marjan | 12266 |
Vir: Osebne bibliografije
in: SICRIS
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