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Little attention has been paid to adrenal sustentacular cells, and several major histology textbooks do not even describe them. This study presents a detailed morphological description of sustentacular cells using immunolight microscopy and an antibody against brain-type fatty acid-binding protein. The immunopositive sustentacular cells and processes formed lattices with holes of various sizes and compactnesses or openness. In addition, weakly immunostained sheet-like structures with ill-defined contours were often associated with the processes and lattices. In the carotid body, which has traditionally been classified under the name of paraganglia in common with the adrenal medulla, immunostained sustentacular cell processes formed lattices in association with the weakly immunostained sheet-like structures, but the lattices with sheets were more compact and rigid than the adrenal medulla, and appeared like individually distinct compartments. In the ganglion, the immunostained satellite cell processes with the sheets tightly enclosed individual neurons. As a result, the immunostained sheet-like structures were regarded as en-face views of thinly flattened sustentacular cytoplasmic envelopes partially covering the chromaffin cells in the adrenal medulla, and widely in the carotid body in a way rather similar to the satellite cells in the ganglion. In brief, the terminal enclosing portions of adrenal sustentacular cell processes, in cut-views, were too thin/flat to be recognized as distinct lines in immuno-light microscopy because of its resolution limit. They are recognized in en-face views as entities of a substantially spacious extension in immuno-light microscopy.

The present immunohistochemical study was attempted to localize in the submandibular glands of mice at various postnatal stages a diacylglycerol kinase (DGK) isoform termed DGKζ which is characterized by a nuclear localization signal and a nuclear export signal. This attempt was based on following facts: the continuous postnatal differentiation of glandular cells in the rodent submandibular gland, the regulatory role of DGK in the activity of protein kinase C (PKC) through attenuation of diacylglycerol (DAG), and the possible involvement of PKC in various cellular activities including the saliva secretion as well as the cell differentiation. As a result, a selective localization of immunoreactivity for DGKζ was detected in terminal tubule (TT) cells which comprise a majority of the newborn acinar structure and differentiate into the intercalated duct cells and/or the acinar cells. The immunoreactivity was deposited in portions of the cytoplasm lateral and basal to the nucleus, but not in the nuclei themselves. Although the immunoreactive TT cells remained until later stages in female specimen than in male, they eventually disappeared in both sexes by young adult stages. The present finding suggests that the regulatory involvement of DGKζ in PKC functions via control of DAG is exerted in the differentiation of the TT cells. In addition, another possible involvement of DGKζ in the regulation of secretion of the TT cells as well as its functional significance of its nuclear localization in the submandibular ganglion cells was also discussed.

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It is known that phosphatidylinositol phosphate 5 kinase (PIP5K) γ and phospholipase C (PLC) β3, working sequentially in the phosphoinositide cycle, are localized in dorsal root ganglion (DRG) somata and are involved in the regulation of pain and related sensations. However, the sites of their involvement have remained to be clarified. In the present study, immunoreactivity for PLCβ3 was distinct only in the central process of mouse DRG, but not in its peripheral process, in contrast to distinct PIP5Kγ‐immunoreactivity in both peripheral and central DRG processes. No nerve terminals showing immunoreactivity for PLCβ3 were detected in any peripheral sensory fields, similar to PIP5Kγ‐immunoreactivity. In DRG somata, PIP5Kγ‐immunoreactivity was rather confined to the neurolemma in which dots and threads were discerned in 3D bright field light microscopy. This feature well corresponded to its discontinuous localization along the plasma membranes in immuno‐electron microscopy. In contrast, PLCβ3‐immunoreactivity occurred diffusely throughout the somata, but did not take distinct appearance of immunoreaction on neurolemma or plasma membranes, unlike PIP5Kγ‐immunoreactivity. In addition, satellite glial cells were immunonegative for PLCβ3, but immunopositive for PIP5Kγ. The involvement of PLCβ3 in regulation of pain and related sensations is thus suggested to be mainly exerted at levels of the DRG soma and its upstream, but to be less significant in the peripheral sensory fields, similar to PIP5Kγ. The possibility is also suggested that PIP, PIP5Kγ‐target, is localized heterogeneously, but PIP2, PLCβ3‐target, is localized homogenously over the plane of the neuronal plasma membranes. Research Highlights PIP5Kγ, different from PLCβ3, was localized heterogeneously on neuronal membranes, and this difference was demonstrated in 3D‐bright field immuno‐light and electron microscopy. Either PIP5Kγ or PLCβ3 was not detected in peripheral nerve terminals.

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.

SUMMARY:According to recent studies, it is highly possible that the occurrence of vesicular inhibitory amino acid transporter (VIAAT) is a good marker of GABA-signaling not only in the brain, but also in extra-brain tissue cells containing GABA and GAD. In view of this, the present study was attempted to localize VIAAT-immunoreactivity in the submandibular gland of mice. In the present study, the submandibular glands of male mice at various postnatal developmental stages were examined for detailed localization of VIAAT-immunoreactivity in immunohistochemistry at light microscopic level. The immunoreactivity for VIAAT was localized in epithelial cells of proximal and distal excretory ducts with the striated portion more intensely immunopositive at young postnatal stages. No significant immunoreactivity was seen in the acinar cells throughout the postnatal development. In addition, the immunoreactivity for VIAAT was detected in the salivary parasympathetic ganglionic neurons, but not in any nerve fibers surrounding the glandular cells. Furthermore, VIAAT-immunoreactivity was found in smooth muscle cells forming the outermost layer of intralobular arterioles. From the present findings, it is possible that GABA plays roles as paracrine and autocrine regulators in the saliva secretion as well as the gland development.

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