Expression and localization of endogenous phospholipase Cß3 confined to basal cells in situ of immature ducts and adult excretory ducts of submandibular gland of mice.

Our previous study demonstrated that, different from the parotid and sublingual glands, the submandibular glands of adult mice did not show an immunoblot band for PLCß3 which is critical in the secretion mechanism by muscarinic cholinergic signaling. Therefore, the submandibular glands of mice at various stages of postnatal development were examined for this enzyme molecule in immunoblot and immunohistochemistry. In immunoblot, a weak band for PLCß3-expression was detected only at early postnatal stages. In immunohistochemistry, PLCß3-immunoreactivity was distinctly found in most basally located cells of immature ducts, while the immunoreactivity was weakly seen in terminal tubule cells without significant immunoreactivity in adjacent acinar cells. In contrast, the immunoreactivity was distinctly found in some basal cells of adult excretory ducts, and it was ultrastructurally localized densely in close association with bundles of tonofilaments in the cells. The present finding suggests the possibility that Ca2+ signaling governed by phospholipase Cß3 is involved in the differentiation of ductal basal cells into apical cells through control of keratin molecule(s) in the cells.

Localization of phospholipase C ß3 in the major salivary glands of adult mice.

Phospholipase C (PLC)ß has a role in saliva secretion by controlling intracellular Ca2+via its product, IP3. The present study was attempted to localize PLCß isoforms in mouse salivary glands in situ. A single major band was detected for PLCß3 in immunoblots of the parotid and sublingual glands (PG, SLG), while no such band was seen in the submandibular gland (SMG). No bands were detected for PLCß1 or 4 in the three glands. In immuno-light microscopy of PG and SLG, substantial immunoreactivity for PLCß3 was seen in the cytoplasm including the plasmalemma of almost all ductal cells, while no distinct immunoreactivity was discerned in most acinar cells except for sublingual demilune cells. Numerous ductal cells exhibited higher immunoreactivity for PLCß3 in their apical/supranuclear cell domain including the plasmalemma than in the basal/infranuclear domain, indicating an apico-basal polarity. In immuno-gold electron microscopy of PG ducts and SLG ducts and demilunes, most gold particles were found in association with plasma membranes as well as various intracellular membranes, most of which formed small oblong or flattened vesicles and vacuoles. A few particles were seen without association with any membranous structures. The present finding supports the previous physio-pharmacological result that Ca2+-signaling proteins as well as initial intracellular Ca2+ changes occur in the apical cell domain including the plasma membranes of the exocrine cells.

Localization of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) α, ß, γ in the three major salivary glands in situ of mice and their response to ß-adrenoceptor stimulation.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), which is composed of three isozymes (α, ß and γ), catalyzes the production of phosphatidylinositol bisphosphate (PIP2). This phospholipid functions in membrane trafficking, as an anchor for actin cytoskeletons and as a regulator of intramembranous channels/transporters. It is also a precursor of such second messengers as diacylglycerol, inositol triphosphate and phosphatidylinositol (3,4,5)-triphosphate. In the present study, the expression and localization of endogenous PIP5Ks were examined in the three major salivary glands of young adult mice in situ. In western blotting of normal control glands, immunoreactive bands for individual PIP5Ks were detectable, with the highest density in the parotid gland and the weakest density in the submandibular gland. In immuno-light microscopy under non-stimulated condition, weak immunoreactivity for PIP5Kα was confined to the apical plasmalemma in parotid, but not sublingual or submandibular, acinar cells. Immunoreactivity for PIP5Kß was weak to moderate and confined to ductal cells but not acinar cells, whereas that for PIP5Kγ was selectively and intensely detected in myoepithelial cells but not acinar cells, and it was weak in ductal cells in the three glands. In western blot of the parotid gland stimulated by isoproterenol, a ß-adrenoceptor agonist, no changes were seen in the intensity of immunoreactive bands for any of the PIP5Ks. In contrast, in immuno-light microscopy, the apical immunoreactivity for PIP5Kα in parotid acinar cells was transiently and distinctly increased after the stimulation. The increased immunoreactivity was ultrastructurally localized on most apical microvilli and along contiguous plasma membrane, where membranous invaginations of various shapes and small vesicles were frequently found. It was thus suggested that PIP5Kα is involved in post-exocytotic membrane dynamics via microvillous membranes. The present finding further suggests that each of the three isoforms of PIP5K functions through its product PIP2 discretely in different cells of the glands to regulate saliva secretion.

Heterogeneous localization of muscarinic cholinoceptor M1 in the salivary ducts of adult mice.

We hypothesize variation in expression and localization, along the course of the glandular tubule, of muscarinic cholinergic receptor M1 which plays as a distinct contribution, though minor in comparison with M3 receptor, in saliva secretion. Localization of the M1 receptor was examined using immunohistochemistry in three major salivary glands. Although all glandular cells were more or less M1-immunoreactive, acinar cells were weakly immunoreactive, while ductal cells exhibited substantial M1-immunoreactivity. Many ductal cells exhibited clear polarity with higher immunoreactivity in their apical/supra-nuclear domain. However, some exhibited indistinct polarity because of additional higher immunoreactivity in their basal/infra-nuclear domain. A small group of cells with intense immunoreactivity was found, mostly located in the intercalated ducts or in portions of the striated ducts close to the intercalated ducts. In immuno-electron microscopy, the immunoreactive materials were mainly in the cytoplasm including various vesicles and vacuoles. Unexpectedly, distinct immunoreactivity on apical and basal plasma membranes was infrequent in most ductal cells. The heterogeneous localization of M1-immunoreactivity along the gland tubular system is discussed in view of possible modulatory roles of the M1 receptor in saliva secretion.

Expression and localization of VIAAT in distal uriniferous tubular epithelium of mouse.

Vesicular inhibitory amino acid transporter (VIAAT) is a transmembrane transporter which is responsible for the storage of gamma-aminobutyric acid (GABA) or glycine in synaptic vesicles. According to recent studies, GABA is known to be expressed in the kidney. For clear understanding of the intra-renal GABA signaling, the localization of VIAAT was examined in the present study. Intense immunoreactivity was found largely confined to the distal tubule epithelia, especially distinct in the inner medulla, although the immunoreactivity was discerned more or less in all tubules and glomeruli. No distinct immunoreactivity was seen in capillary endothelia or interstitial fibroblasts. In immuno-DAB and immuno-gold electron microscopy, the immunoreaction was found at the basal infoldings of plasma membranes and basal portions of the lateral plasma membranes, but not in any vesicles or vacuoles within the distal tubular cells. The significance of the enigmatic finding, localization of a vesicular molecule on selected portions of the plasma membrane of distal tubular cells, was discussed in view of the possibility of paracrine or autocrine effects of GABA on some other uriniferous tubular cells or interstitial cells.

Ultrastructural histometric evidence for expansion of the sustentacular cell envelope in response to hypersecretion of adrenal chromaffin cells in mice.

In our previous immuno-light microscopic study with an antibody for fatty acid binding protein of type 7 or brain type (FABP-7, B-FABP), the adrenomedullary sustentacular cells were revealed to have secondary processes that present faint immunostaining and an ill-defined sheet-like appearance, in addition to the well-recognized primary processes that present distinct immunostaining and a fibrous appearance. The secondary processes were regarded as corresponding to known ultrastructural profiles of sustentacular cells with a thickness of less than 0.2 µm (the resolution limit of light microscopy), and the processes were considered to be largely responsible for enveloping chromaffin cells. Due to those findings, the present immuno-electron microscopic study was performed to determine whether the secondary processes change the extent of their envelope for chromaffin cells under the intense secretion induced by water immersion-restraint stress. To achieve this, we focused on immunopositive ultrastructural profiles with a thickness of less than 0.2 µm. The measured lengths of the immunopositive profiles in the specimens from stressed mice were found to be significantly larger than those in specimens from normal mice, indicating an increase in the extent of the envelope of the sheet-like processes for the chromaffin cells. Thus, confining our measurements to the secondary process profiles, not the entire cell profiles, proved to be a key factor in the detection-for the first time-of the change in size of the sustentacular cell envelope upon changes in the secretory activity of enveloped chromaffin cells. The possible functional significance of this change in size is discussed here.

Immunohistochemical localization of cannabinoid receptor 1 (CB1) in the submandibular gland of mice under normal conditions and when stimulated by isoproterenol or carbachol.

OBJECTIVE: We wished to investigate the subcellular localization of CB1, a receptor for the endocannabinoids in mouse submandibular glands (SMGs) under normal conditions and when stimulated by adrenergic or cholinergic agonists. MATERIALS AND METHODS: SMGs of both male and female adult mice were utilized for immunoblotting and immuno-light and -electron microscopic analyses. Isoproterenol and carbachol were used as adrenergic and cholinergic stimulants, respectively. SMGs were examined at 15, 30, 60 and 120min after intraperitoneal injection of these agents. RESULTS: Selective localization of intense immunoreactivity for CB1 in the granular convoluted ductal cells was confirmed by immunoblotting and the antigen absorption test. In SMGs of control male mice, CB1-immunoreactivity was evident on the basolateral plasma membranes, including the basal infoldings, but was absent on the apical membranes in the ductal cells. Localization and intensity of CB1-immunoreactivity were essentially the same in SMGs of female mice. The immunoreactivity was transiently localized in the apical plasmalemma of some acinar and granular ductal cells of male SMGs shortly after stimulation by isoproterenol, but not by carbachol. CONCLUSION: The present finding suggests that CB1 functions primarily in the basolateral membranes of the granular convoluted ductal cells of SMGs under normal conditions, and that the CB1 can function additionally in the apical membrane of acinar and granular ductal cells for modulation of the saliva secretory condition via adrenoceptors.