Characterization of Ryanodine-sensitive Ca2+ Release from Microsomal Vesicles of Rat Parotid Acinar Cells: Regulation by Cyclic ADP-ribose

We have measured ryanodine (caffeine)-sensitive 45Ca2+ release from isolated microsomal vesicles of endoplasmic reticulum prepared from rat parotid acinar cells. After a steady state of ATP-dependent 45Ca2+ uptake, the addition of caffeine (40 mm), ryanodine (10∼500 μm) or an NAD+ metabolite, cyclic ADP-ribose (cADPR, 4 μm) released about 10% of the 45Ca2+ that had been taken up. The 45Ca2+ release was not inhibited by heparin, an antagonist of IP3 receptor. The effects of caffeine, ryanodine and cADPR on 45Ca2+ release were also tested in the presence of thapsigargin (TG), an inhibitor of microsomal Ca2+-ATPase. When caffeine (10∼40 mm), ryanodine (10 μm) or cADPR (1∼10 μm) was added in the medium with 100 nm TG, a significant 45Ca2+ release was seen, while higher concentrations of ryanodine (>100 μm) did not cause any 45Ca2+ release in the presence of TG. The initial rate of caffeine (40 mm)-induced 45Ca2+ release was increased by a pretreatment with 10 μm ryanodine, whereas the caffeine-induced 45Ca2+ release was strongly inhibited by the presence of a higher concentration (500 μm) of ryanodine. cADPR-induced 45Ca2+ release was also inhibited by 500 μm ryanodine. Caffeine (40 mm)- or cADPR (4 μm)-induced 45Ca2+ release was abolished by a presence of ruthenium red (50∼100 μm). The presence of a low concentration (0.5 μm) of cADPR shifted the dose-response curve of caffeine-induced 45Ca2+ release to the left. These results indicate the presence of a ryanodine sensitive Ca2+ release mechanism in the endoplasmic reticulum of rat parotid acinar cells that is distinct from the IP3-sensitive Ca2+ channel and is activated by caffeine, cADPR and a low concentration (10 μm) of ryanodine, but is inhibited by higher concentrations (>100 μm) of ryanodine and ruthenium red. The properties of the ryanodine-sensitive mechanism are similar to that of the ryanodine receptor as described in muscle cells.