The presence and regulation of the ryanodine receptor in the pancreatic acinar cell

The regulation of cytosolic Ca{dollar}sp{lcub}2+{rcub}{dollar} is important for a variety of cell functions. Inositol 1,4,5trisphosphate (IP{dollar}sb3){dollar} is a well characterized activator of Ca{dollar}sp{lcub}2+{rcub}{dollar} release from internal Ca{dollar}sp{lcub}2+{rcub}{dollar} stores. Two non-IP{dollar}sb3{dollar} compounds that may regulate Ca{dollar}sp{lcub}2+{rcub}{dollar} in the pancreatic acinar cell are acyl-Coenzyme A (CoA) and cyclic ADP-ribose (cADPR). To study the role of non-IP{dollar}sb3{dollar} regulated Ca{dollar}sp{lcub}2+{rcub}{dollar} release, we measured the presence and effect of acyl-CoA and cADPR on Ca{dollar}sp{lcub}2+{rcub}{dollar} movements and we looked for the presence of the ryanodine receptor, an IP{dollar}sb3{dollar}-insensitive Ca{dollar}sp{lcub}2+{rcub}{dollar} channel. Both palmitoyl-CoA and cADPR caused Ca{dollar}sp{lcub}2+{rcub}{dollar} release from isolated and permeablized acinar cells. The palmitoyl-CoA-sensitive pool was distinct from, and overlapping with the IP{dollar}sb3{dollar}-sensitive Ca{dollar}sp{lcub}2+{rcub}{dollar} pool. The effects of submaximal doses of IP{dollar}sb3{dollar} or cyclic ADP-ribose plus palmitoyl-CoA were additive. Ryanodine and caffeine or elevated resting (Ca{dollar}sp{lcub}2+{rcub}{dollar}) sensitized the Ca{dollar}sp{lcub}2+{rcub}{dollar} pool to the actions of palmitoyl-CoA. Acyl-CoA levels in pancreatic acini were measured by separation, and quantification using reverse phase HPLC, and were found to be 10.17 {dollar}pm{dollar} 0.93 nmol/mg protein. cADPR was isolated and purified from acinar cells by extraction and sequential separation using TLC and HPLC. The purified compound from acini was found to release Ca{dollar}sp{lcub}2+{rcub}{dollar} by bioassay and the Ca{dollar}sp{lcub}2+{rcub}{dollar} release was desensitized by authentic cADPR. To determine whether or not the ryanodine receptor is present in the pancreatic acinar cell, we performed RT-PCR, Western blot and functional analysis on pancreatic acinar cells. RT-PCR products from pancreatic acini showed the presence of RNA for RyR types 1, 2, and 3. These products were of the expected size, as were the endonuclease restriction digest products. Western blot analysis of acinar cell immunoprecipitates showed a high MW protein band that was much less intense when immunoprecipitated in the presence of ryanodine receptor peptide. This report shows palmitoyl-CoA and cADPR are present in and cause Ca{dollar}sp{lcub}2+{rcub}{dollar} release from pancreatic acinar cells and that pancreatic acinar cells have multiple forms of functional ryanodine receptors. In addition, we suggest the presence of an IP{dollar}sb3{dollar}-insensitive, palmitoyl-CoA sensitive Ca{dollar}sp{lcub}2+{rcub}{dollar} store in pancreatic acinar cells and suggest that palmitoyl-CoA may be needed for Ca{dollar}sp{lcub}2+{rcub}{dollar}-induced Ca{dollar}sp{lcub}2+{rcub}{dollar} release.