Organelles containing inositol trisphosphate receptor type 2 in adrenal medullary cells.

To identify which organelles contained inositol trisphosphate (InsP(3)) receptor type 2 (InsP(3)R2) in adrenal medullary (AM) cells, immunocytochemical and biochemical studies were performed on AM cells of several species. InsP(3)R2-like immunoreactive materials produced by two different anti-InsP(3)R2 antibodies (Abs) (Chemicon and Sigma) were distributed in rat AM cells in agreement with BODIPY-FL-InsP(3) binding sites. For two other Abs (KM1083 and Santa Cruz), some of the anti-InsP(3)R2 immunoreactive materials were stained with an anti-dopamine-beta-hydroxylase Ab, but not by BODIPY-FL-InsP(3). BODIPY-FL-thapsigargin binding sites were consistent with a distribution of the endoplasmic reticulum (ER) identified by an anti-calnexin Ab, and a prior application of thapsigargin significantly eliminated BODIPY-FL-thapsigargin bindings, suggesting that BODIPY-FL-thapsigargin bindings were mediated by thapsigargin, but not the fluorescence molecule. The anti-InsP(3)R2 Ab that produced stainings consistent with BODIPY-FL-InsP(3) bindings recognized a protein with about 250 kDa. A fractional analysis of bovine adrenal medullae revealed that the 250 kDa InsP(3)R2 was detected in a crude membrane fraction, but not in a secretory granule fraction. The results suggest that the InsP(3)R2 was present in the ER, but not in secretory granules in AM cells.