In situ activation of the type 2 ryanodine receptor in pancreatic beta cells requires cAMP-dependent phosphorylation (Islets of Langerhansycaffeineycalcium)

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 Ca 21 from the endoplasmic reticulum (ER) in the form of small spikes in only 14% of cultured fura-2 loaded beta cells from obyob mice. Surprisingly, when forskolin, an activator of adenylyl cy- clase was present, caffeine induced larger Ca 21 spikes in as many as 60% of the cells. Forskolin or the phosphodiesterase-resistant PKA activator Sp-cAMPS alone did not release Ca 21 from ER. 4-Chloro-3-ethylphenol (4-CEP), an agent that activates RYs in other cell systems, released Ca 21 from ER, giving rise to a slow and small increase in (Ca 21 )i in beta cells. Prior exposure of cells to forskolin or caffeine (5 mM) qualitatively altered Ca 21 release by 4-CEP, giving rise to Ca 21 spikes. In glucose-stimulated beta cells forskolin induced Ca 21 spikes that were enhanced by 3,9- dimethylxanthine, an activator of RYs. Analysis of RNA from islets and insulin-secreting bTC-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. Ryanodine receptors (RY) are Ca 21 channels in the endo-