Apical Vesicles Bearing Inositol 1,4,5-trisphosphate Receptors in the Ca²⁺Initiation Site of Ductal Epithelium of Submandibular Gland

In polarized epithelial cells, agonists trigger Ca²⁺ waves and oscillations. These patterns may be caused by the compartmentalization of inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ pools into specific regions. We have investigated the relationship between the distribution of IP₃ receptors (IP₃Rs) and the spatiotemporal pattern of Ca²⁺ signaling in the duct cells of the rat submandibular gland (SMG). Using immunofluorescence, although labeling was somewhat heterogeneous, the IP₃Rs were colocalized to the apical pole of the duct cells. Immunoelectron microscopy identified small apical vesicles bearing IP₃R2 in some types of duct cells. Real-time confocal imaging of intact ducts demonstrated that, after carbachol stimulation, an initial Ca²⁺ spike occurred in the apical region. Subsequently, repetitive Ca²⁺ spikes spread from the apical to the middle cytoplasm. These apical Ca²⁺ initiation sites were found only in some “pioneer cells,” rather than in all duct cells. We performed both Ca²⁺ imaging and immunofluorescence on the same ducts and detected the strongest immunosignals of IP₃R2 in the Ca²⁺ initiation sites of the pioneer cells. The subcellular localization and expression level of IP₃Rs correlated strongly with the spatiotemporal nature of the intracellular Ca²⁺ signal and distinct Ca²⁺ responses among the rat SMG duct cells.