Expression with early postnatal peak and female-dominant sexual dimorphism of phospholipase D (PLD) 2 in submandibular gland ducts in situ of mice.

In the present study, expression and localization of PLD2 were examined in mouse submandibular glands in situ in comparison with those of PLD1 previously reported by the present authors. In immunoblots, PLD2-expression was high at postnatal 0 week (P0W) and P2W, and at P4W decreased considerably with the decrease of the male gland more marked, and at P8W it was undetectable in the male gland, but remained faint in the female. In the male gland at P8W after castration at P6W, PLD2-expression reappeared faintly. In the female glands with daily injections of testosterone started at P6W, at P8W the expression was undetectable. In immunohistochemistry, PLD2 was localized throughout immature duct epithelia without distinct regional differences in its intensity at P0W and P2W. PLD2-localization was weak and diffuse in granular convoluted tubules at P4W and negligible there at P8W in the female gland, while it was at negligible levels in the tubules at P4W and at undetectable levels there at P8W in the male gland. The expression changes detected in immunoblots after castration or testosterone injection were duplicated in granular convoluted tubules in immunohistochemistry. The present findings suggest that PLD2 is dominantly involved in proliferation/differentiation/apoptosis of most immature ductal cells at early postnatal and at least perinatal stages, different from PLD1. This also indicates that the female-dominant sexual dimorphism of PLD2-expression occurs in synchrony of differentiation of granular convoluted tubules at puberty and at young adulthood, similar to PLD1 though at much lower levels.

Expression of endogenous phospholipase D1, localized in mouse submandibular gland, is greater in females and is suppressed by testosterone.

To clarify the signal transduction mechanism in the differentiation and secretion of salivary glandular cells, the present study was attempted to examine in the submandibular gland (SMG) of mice, the expression and localization of phospholipase D1 (PLD1), one of the important effector molecules working in response to the activation of intramembranous receptors by first messengers. In immunoblotting analysis, the expression of PLD1 was high at postnatal 4 weeks (P4W) and decreased at P8W, and it was at negligible levels at newborn stage (P0W) and postnatal 2 weeks (P2W). The expression of PLD1 was greater in females, and it was suppressed by administration of testosterone to female mice. In immuno-light microscopy, immunoreactivity for PLD1 at P4W was moderate to intense, in the forms of dots and globules mainly in the apical domains of immature granular convoluted tubule (GCT)-cells localized largely in the proximal portion of the female GCT. By P8W, it decreased in intensity and remained weak to moderate along the apical plasmalemma of cells throughout the course of the female GCT, whereas it was faint throughout the GCT of the male SMG at P4W and negligible at P8W. In immuno-electron microscopy, immature GCT-cells characterized by electron-lucent granules were immunoreactive and the immunoreactive materials were deposited close to, but not within, those granules. Typical GCT cells, characterized by electron-dense granules, were immunonegative. No significant immunoreaction for PLD1 was seen in acini of SMGs of either sex at any time point examined. It is suggested that PLD1 is involved in the signaling for secretion of immature GCT cells and influences differentiation of these cells, probably through their own secretory substances.

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.