Abstract
InsP3Rs were reported to be present not only in the ER, but also in secretory granules in bovine adrenal chromaffin cells. Thus, we investigated immunocytochemically which organelles contained InsP3Rs in rat adrenal chromaffin cells. The distribution of BODIPY-InsP3, which is a InsP3 conjugated with a fluorescent molecule, was consistent with that of the ER identified by using an anti-calnexin Ab. Anti-InsP3R type 2 Abs of Sigma and Chemicon each produced immunoreactive materials in agreement with BODIPY-InsP3 binding sites. On the other hand, part of materials immunoreactive to the anti-InsP3R2 mAb (KM1083) at the cell periphery were stained by using an anti-dopamine-β-hydroxylase Ab, but not by BODIPY-InsP3. BODIPY-thapsigargin binding sites were consistent with distribution of the ER identified by the anti-calnexin Ab, and a prior application of thapsigargin significantly eliminated BODIPY-thapsigargin bindings. Application of muscarine failed to induce a Ca2+ mobilization in the presence of thapsigargin, suggesting that muscarine mobilized Ca2+ ions from thapsigarin-sensitive sites. The anti-InsP3R2 Ab of Chemicon recognized a protein with about 250 kDa alone, whereas KM1083 detected an about 200 kDa protein as well as the 250 kDa in a crude membrane fraction of the adrenal medulla. The results indicate that the InsP3R2 is present in the ER, but not in secretory granules in rat adrenal chromaffin cells. [Jpn J Physiol 55 Suppl:S75 (2005)]
