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 rec eptors (RY) in different cells are poorly understood. Here we demonstr ate that caffeine (10 mM) released Ca2+ from the endoplasmic reticulum (ER) in the form of small spikes in only 14% of cultured fura-2 loade d beta cells from ob/ob mice. Surprisingly, when forskolin, an activat or of adenylyl cyclase was present, caffeine induced larger Ca2+ spike s in as many as 60% of the cells, Forskolin or the phosphodiesterase-r esistant PKA activator Sp-cAMPS alone did not release Ca2+ from ER 4-C hloro-3-ethylphenol (4-CEP), an agent that activates RYs in other cell systems, released Ca2+ from ER, giving rise to a slow and small incre ase in [Ca2+](i) in beta cells. Prior exposure of cells to forskolin o r caffeine (5 mM) qualitatively altered Ca2+ release by 4-CEP, giving rise to Ca2+ spikes. In glucose-stimulated beta cells forskolin induce d Ca2+ spikes that were enhanced by 3,9-dimethylxanthine, an activator of RYs, Analysis of RNA from islets and insulin-secreting beta TC3-ce lls 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 require s cAMP-dependent phosphorylation, a process that recruits the receptor in a functionally operative form.