Localization of PIP5Kγ selectively in proprioceptive peripheral fields and also in sensory ganglionic satellite cells as well as neuronal cell membranes and their central terminals

Based on a previous study by others reporting that PIP5Kγ (phosphatidylinositol 4-phosphate 5-kinase γ) and its product, phosphatidylinositol 4,5 bisphosphate (PIP₂), are involved in the regulation of nociception, the present immunohistochemical study examined the localization of PIP5Kγ-immunoreactivity in dorsal root ganglia (DRG) and their peripheral and central terminal fields. PIP5Kγ-immunoreactivity was localized for the first time in the muscle spindles, in which it was found in I-bands of polar regions of intrafusal muscle fibers and also in sensory nerve terminals abutting on equatorial regions of the muscle fibers. This finding indicates the involvement of PIP5Kγ in the proprioception and suggests somehow complicated mechanisms of its involvement because of its heterogeneous localization in intra-I-band structures. In DRG, on the other hand, PIP5Kγ-immunoreactivity was shown to be localized heterogeneously, but not evenly, over apposed plasma membranes of both neurons and ganglionic satellite cells in immune electron microscopy. In addition, no peripheral nerve terminals of DRG showing its distinct immunoreactivity were found in most peripheral fields of nociception and any other sensory perception except for the proprioception through muscle spindles. In contrast, numerous central terminals of DRG in the spinal posterior horn were immunoreactive for it. This finding leads us to consider the possibility that the regulation by PIP5Kγ of nociception is dominantly exerted in DRG and sensory neural tracts central, rather than peripheral, to DRG.     

Localization of EFA6 (exchange factor for ARF6) isoform D in steroidogenic testicular Leydig cells of adult mice

EFA6 (exchange factor for ARF6) activates Arf6 (ADP ribosylation factor 6) by exchanging ADP to ATP and the resulting activated form of Arf6 is involved in the membrane trafficking and actin remodeling of cells. Our previous study has shown the selective expression/localization of EFA6D in steroidogenic adrenocortical cells in situ of adult mice. In view of the previous finding, the present study was undertaken to examine its localization in mouse Leydig cells representing another steroidogenic cell species in order to further support the possible involvement of the EFA6/Arf6 cascade via membrane trafficking in the regulation of steroidogenesis and/or secretion. A distinct band for EFA6D with the same size as that of the brain was detected in the testis of adult mice. In immuno-light microscopy, immunoreactivity for EFA6D was seen throughout the cytoplasm in most Leydig cells without any distinct accumulation along the plasmalemma. Lack of immunoreactivity for EFA6D was seen in the seminiferous tubular epithelium. In immuno-electron microscopy, the immune-labeling was seen in sporadic/focal patterns on plasma membranes and some vesicles and vacuoles subjacent to the plasma membranes. More constant and rather predominant is the labeling on numerous mitochondria. No immuno-labeling was seen in lipid droplets. The present study suggests that EFA6D is somehow involved in regulation of the synthesis and/or secretion of testosterone through the membrane-traffic by activation of Arf6. In addition, EFA6D is suggested to play in mitochondria some yet unidentified roles rather independent of Arf6-activation, which remains to be elucidated.     

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.     

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

Phosphatidylinositol 4‐phosphate 5‐kinase (PIP5K), which is composed of three isozymes (α, β 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 PIP5Kβ 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 β‐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.     

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.     

Impaired induction of cystatin S gene expression by isoproterenol in the submandibular gland of hypophysectomized rats

Cystatin S, an inhibitor of cysteine proteases, is produced and secreted by acinar cells of the rat submandibular gland. Expression of the cystatin S gene is known to be induced at high levels by the beta-adrenergic agonist isoproterenol. In the present study, we revealed that in the submandibular gland of hypophysectomized adult male rats, the levels of induced cystatin S mRNA 24 h after a single administration of isoproterenol are strikingly lower than those in the gland of normal rats. Administration of one of the pituitary-dependent hormones testosterone, estradiol, dexamethasone and thyroxine, together with isoproterenol resulted in marked enhancement of the isoproterenol-induced cystatin S mRNA expression in hypophysectomized rats, whereas administration of any of these hormones alone had no significant effect. These results suggested the existence of cross-talk between the signaling pathways of steroid hormones and isoproterenol in inducing cystatin S gene expression in the rat submandibular gland.     

Localization of phospholipase C β3 in the major salivary glands of adult mice

Phospholipase C (PLC)β has a role in saliva secretion by controlling intracellular Ca²⁺ via its product, IP₃. The present study was attempted to localize PLCβ isoforms in mouse salivary glands in situ. A single major band was detected for PLCβ3 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 PLCβ1 or 4 in the three glands. In immuno-light microscopy of PG and SLG, substantial immunoreactivity for PLCβ3 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 PLCβ3 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.