Interplay of Ca2+ and cAMP signaling in the insulin-secreting MIN6 beta-cell line.
Ca2+ and cAMP are important second messengers that regulate multiple cellular processes. Although previous studies have suggested direct interactions between Ca2+ and cAMP signaling pathways, the underlying mechanisms remain unresolved. In particular, direct evidence for Ca2+-regulated cAMP production in living cells is incomplete. Genetically encoded fluorescence resonance energy transfer-based biosensors have made possible real-time imaging of spatial and temporal gradients of intracellular cAMP concentration in single living cells. Here, we used confocal microscopy, fluorescence resonance energy transfer, and insulin-secreting MIN6 cells expressing Epac1-camps, a biosynthetic unimolecular cAMP indicator, to better understand the role of intracellular Ca2+ in cAMP production. We report that depolarization with high external K+, tolbutamide, or glucose caused a rapid increase in cAMP that was dependent on extracellular Ca2+ and inhibited by nitrendipine, a Ca2+ channel blocker, or 2',5'-dideoxyadenosine, a P-site antagonist of transmembrane adenylate cyclases. Stimulation of MIN6 cells with glucose in the presence of tetraethylammonium chloride generated concomitant Ca2+ and cAMP oscillations that were abolished in the absence of extracellular Ca2+ and blocked by 2',5'-dideoxyadenosine or 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase. Simultaneous measurements of Ca2+ and cAMP concentrations with Fura-2 and Epac1-camps, respectively, revealed a close temporal and causal interrelationship between the increases in cytoplasmic Ca2+ and cAMP levels following membrane depolarization. These findings indicate highly coordinated interplay between Ca2+ and cAMP signaling in electrically excitable endocrine cells and suggest that Ca2+-dependent cAMP oscillations are derived from an increase in adenylate cyclase activity and periodic activation and inactivation of cAMP-hydrolyzing phosphodiesterase.
Department of Medicine, The University of Chicago, Chicago, Illinois 60637, USA.
Luis R
LR
Landa
Mark
M
Harbeck
Kelly
K
Kaihara
Oleg
O
Chepurny
Kajorn
K
Kitiphongspattana
Oliver
O
Graf
Viacheslav O
VO
Nikolaev
Martin J
MJ
Lohse
George G
GG
Holz
Michael W
MW
Roe
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism; D051379:Mice; D011189:Potassium Chloride / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015290:Second Messenger Systems / Q000502:physiology; D019789:Tetraethylammonium / Q000378:metabolism; D014044:Tolbutamide / Q000378:metabolism
D005456:Fluorescent Dyes; D020662:Guanine Nucleotide Exchange Factors; D007004:Hypoglycemic Agents; D007328:Insulin; C116122:RAPGEF3 protein, human; D011993:Recombinant Fusion Proteins; D019789:Tetraethylammonium; D011189:Potassium Chloride; D014044:Tolbutamide; D000242:Cyclic AMP; D005947:Glucose; D002118:Calcium