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 Ca(2+) 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 Ca(2+) spikes in as many as 60% of the cells. Forskolin or the phosphodiesterase-resistant PKA activator Sp-cAMPS alone did not release Ca(2+) from ER. 4-Chloro-3-ethylphenol (4-CEP), an agent that activates RYs in other cell systems, released Ca(2+) from ER, giving rise to a slow and small increase in [Ca(2+)](i) in beta cells. Prior exposure of cells to forskolin or caffeine (5 mM) qualitatively altered Ca(2+) release by 4-CEP, giving rise to Ca(2+) spikes. In glucose-stimulated beta cells forskolin induced Ca(2+) spikes that were enhanced by 3,9-dimethylxanthine, an activator of RYs. Analysis of RNA from islets and insulin-secreting βTC-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 (RY(2)). We conclude that in situ activation of RY(2) in beta cells requires cAMP-dependent phosphorylation, a process that recruits the receptor in a functionally operative form.