In situ activation of the type 2 ryanodine receptor in pancreatic beta cells requires cAMP-dependent phosphorylation.
Molecular mechanisms that regulate in situ activation of ryanodine receptors (RY) in different cells are poorly understood. Here we demonstrate that caffeine (10 mM) released Ca2+ from the endoplasmic reticulum (ER) in the form of small spikes in only 14% of cultured fura-2 loaded beta cells from ob/ob mice. Surprisingly, when forskolin, an activator of adenylyl cyclase was present, caffeine induced larger Ca2+ spikes in as many as 60% of the cells. Forskolin or the phosphodiesterase-resistant PKA activator Sp-cAMPS alone did not release Ca2+ from ER. 4-Chloro-3-ethylphenol (4-CEP), an agent that activates RYs in other cell systems, released Ca2+ from ER, giving rise to a slow and small increase in [Ca2+]i in beta cells. Prior exposure of cells to forskolin or caffeine (5 mM) qualitatively altered Ca2+ release by 4-CEP, giving rise to Ca2+ spikes. In glucose-stimulated beta cells forskolin induced Ca2+ spikes that were enhanced by 3,9-dimethylxanthine, an activator of RYs. Analysis of RNA from islets and insulin-secreting betaTC-3-cells by RNase protection assay, using type-specific RY probes, revealed low-level expression of mRNA for the type 2 isoform of the receptor (RY2). We conclude that in situ activation of RY2 in beta cells requires cAMP-dependent phosphorylation, a process that recruits the receptor in a functionally operative form.
Islam
M S
MS
The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, S-171 76 Stockholm, Sweden.
Leibiger
I
I
Leibiger
B
B
Rossi
D
D
Sorrentino
V
V
Ekström
T J
TJ
Westerblad
H
H
Andrade
F H
FH
Berggren
P O
PO
D000818:Animals; D001692:Biological Transport; D002110:Caffeine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D000697:Central Nervous System Stimulants / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism*; D051379:Mice; D008820:Mice, Obese; D010766:Phosphorylation; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
D000697:Central Nervous System Stimulants; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D000242:Cyclic AMP; D002118:Calcium