Kenichiro Kikuchi

Interaction between transcellular and paracellular water transport pathways through Aquaporin 5 and the tight junction complex

To investigate potential physiological interactions between the transcellular and paracellular pathways of water transport, we asked whether targeted deletion of Aquaporin 5 (AQP5), the major transcellular water transporter in salivary acinar cells, affected paracellular transport of 4-kDa FITC-labeled dextran (FITC-D), which is transported through the paracellular but not the transcellular route. After i.v. injection of FITC-D into either AQP5 wild-type or AQP5−/− mice and saliva collection for fixed time intervals, we show that the relative amount of FITC-D transported in the saliva of AQP5−/− mice is half that in matched AQP5+/+ mice, indicating a 2-fold decrease in permeability of the paracellular barrier in mice lacking AQP5. We also found a significant difference in the proportion of transcellular vs. paracellular transport between male and female mice. Freeze-fracture electron microscopy revealed an increase in the number of tight junction strands of both AQP5+/+ and AQP5−/− male mice after pilocarpine stimulation but no change in strand number in female mice. Average acinar cell volume was increased by ≈1.4-fold in glands from AQP5−/− mice, suggesting an alteration in the volume-sensing machinery of the cell. Western blots revealed that expression of Claudin-7, Claudin-3, and Occludin, critical proteins that regulate the permeability of the tight junction barrier, were significantly decreased in AQP5−/− compared with AQP5+/+ salivary glands. These findings reveal the existence of a gender-influenced molecular mechanism involving AQP5 that allows transcellular and paracellular routes of water transport to act in conjunction.

The Structure of Tight Junctions in Mouse Submandibular Gland

Salivary gland cells are joined by junctional complexes consisting of a tight junction (TJ), zonula adherens and one or more desmosomes. TJs regulate paracellular permeability, maintain separate apical and basolateral membrane domains, and serve as signaling centers. We examined TJs of mouse submandibular glands (SMG) in thin sections and freeze‐fracture replicas. TJs between acinar cells and between intercalated duct cells had 2–6 parallel strands on the protoplasmic fracture face, with occasional branches, interconnections and free ends, and corresponding grooves on the extracellular face. Granular duct cell TJs had 2–30 strands, a depth of ≤0.5 μm, and occasional loops extending further basally. Where 3 or 4 cells met, the TJs extended basally ≤1 μm and consisted of 2 parallel boundary strands into which the apical strands inserted. Quantitative analyses showed significant differences in TJ complexity, measured by fractal geometry, and strand number of acinar compared to granular duct cells, and a greater number of strands in male compared to female granular ducts. Pilocarpine stimulation increased TJ strand number in female acinar cells, and increased complexity of male granular duct cell TJs. As the salivary gland water channel aquaporin 5 (AQP5) has been proposed to functionally interact with TJs to regulate salivary fluid composition, we also studied glands from AQP5 knock‐out mice. In males lacking AQP5, granular duct TJs were more complex than those of wild‐type mice, and exhibited more strands following pilocarpine stimulation. The results demonstrate specific gender, cell type and genetic differences in TJ structure and response to stimulation. Anat Rec, 2010.

Uptake of cationized ferritin by the epithelium of the main excretory duct of the rat submandibular gland

Previous studies demonstrated that the main excretory duct (MED) of the rat submandibular gland can internalize exogenous protein in addition to reabsorbing and secreting electrolytes. However, more precise studies have not been conducted. The aim of this study was to elucidate the cell types responsible for endocytosis of an exogenous protein (ferritin) and to follow the movements of the endocytosed protein in the ductal epithelial cells.

Morphological Changes in the Rat Sublingual Gland Parenchyma with Aging

Background: The characteristics of mucous cells in the aging rat sublingual gland were investigated in this study. Particular attention was paid to accumulated amyloid protein and changes of the properties of the secretory granules at the histochemical and ultrastructural level. Objective: This study was designed to examine age-related morphological changes in the sublingual gland of male Wistar rats from 12 to 27 months. Methods: For light microscopy, the sublingual glands were fixed with 10% neutral-buffered formalin, embedded in paraffin, and processed for Alcian blue, Congo red, and TUNEL staining. For transmission electron microscopy, some of the samples were fixed with Karnovsky solution, postfixed with 2% osmium tetroxide, and embedded in epoxy resin for pronase treatment. Results: The sublingual gland showed slight shrinkage after 21 months. After 24 months, Congo red staining showed positive reaction to the intralobular connective tissue surrounding the terminal portions and to the interlobular connective tissue around the blood vessels and the excretory ducts. At 27 months, some of the granules in the serous demilunes had difficulty in digesting with pronase treatment. The appearance rate of TUNEL-positive cells was low in both mucous and serous portions during the observation period, though the positive cell number was higher in the serous than in the mucous portion. Conclusions: These findings indicate that the rat sublingual gland accumulates amyloid protein in the parenchyma and changes the properties of secretory granules of the acinar cells in the serous demilune with aging, though apoptosis of the parenchymal cells and the decrease of the gland weight are slight.

Fine structure of transforming-type granules in mucous cells in the early postnatal rat parotid gland when processed by rapid freezing followed by freeze-substitution fixation.

The present study was designed to clarify the more precise ultrastructural feature of granules, especially mucous granules in the early postnatal rat parotid gland by using rapid freezing followed by freeze‐substitution fixation. The parotid gland of Wistar rats (aged 0–10 days) was removed under anesthesia and immediately underwent cryofixation followed by substitution with osmium tetroxide. After fixation, the samples were embedded in Epon‐Araldite, cut into ultrathin section, and then examined by transmission electron microscopy. Electron microscopy showed that the mucous granules of samples treated by freeze‐substitution fixation had low electron density and were almost spherical in shape with a clear limiting membrane. By Day 5, granules that were a little more electron dense than mucous granules, granules with a more electron dense portion at their periphery, and heterogeneous granules with an internal highly electron dense portion were found. Moreover, cells containing both homogeneous granules with a high electron density similar to that of mature serous granules and heterogeneous granules were observed. These findings demonstrated that the morphology of the transforming‐type mucous granules by chemical fixation in the previous study was an artifact and, as a result, strongly suggested that on the sequential morphological changes of transitional mucous/serous granules by rapid freezing method in the present study, the mucous cells in the early postnatal rat parotid gland undergo transformation to serous cells. Anat Rec 260:387–391, 2000.