Extracellular calcium influx activates adenylate cyclase 1 and potentiates insulin secretion in MIN6 cells

Tetsuya Kitaguchi, Manami Oya, Yoshiko Wada, Takashi Tsuboi, Atsushi Miyawaki

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43 Citations (Scopus)

Abstract

Intracellular cAMP and Ca2+ are important second messengers that regulate insulin secretion in pancreatic β-cells; however, the molecular mechanism underlying their mutual interaction for exocytosis is not fully understood. In the present study, we investigated the interplay between intracellular cAMP and Ca2+ concentrations ([cAMP]i and [Ca2+ ]i respectively) in the pancreatic β-cell line MIN6 using total internal reflection fluorescence microscopy. For measuring [cAMP]i, we developed a genetically encoded yellow fluorescent biosensor for cAMP [Flamindo (fluorescent cAMP indicator)], which changes fluorescence intensity with cAMP binding. Application of high-KCl or glucose to MIN6 cells induced the elevation of [cAMP]i and exocytosis. Furthermore, application of an L-type Ca2+ channel agonist or ionomycin to induce extracellular Ca2+ influx evoked the elevation of [cAMP]i,whereas application of carbachol or thapsigargin, which mobilize Ca2+ from internal stores, did not evoke the elevation of [cAMP]i. We performed RT (reverse transcription)-PCR analysis and found that Ca2+-sensitive Adcy1 (adenylate cyclase 1) was expressed inMIN6 cells. Knockdown of endogenous ADCY1 by small interference RNA significantly suppressed glucose-induced exocytosis and the elevation of both [cAMP]i and [Ca2+ ]i. Taken together, the findings of the present study demonstrate that ADCY1 plays an important role in the control of pancreatic β-cell cAMP homoeostasis and insulin secretion.

Original languageEnglish
Pages (from-to)365-373
Number of pages9
JournalBiochemical Journal
Volume450
Issue number2
DOIs
Publication statusPublished - 2013 Mar 1

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Keywords

  • Calcium
  • CAMP
  • Exchange protein directly activated bycAMP1 (Epac1)
  • Exocytosis
  • Fluorescence microscopy
  • Insulin
  • Total internal reflection
  • Yellow fluorescent protein

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

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