Electron-translucency and partial defects of synaptic basal lamina in the electrocyte synapse of an electric ray (Narke japonica) in 3D embedment-free section electron microscopy.

The synaptic basal lamina of the electrocytes was disclosed to be electron-translucent to some extent when viewed in an en-face direction in embedment-free section transmission electron microscopy (EFS-TEM), and synaptic vesicles located close to the presynaptic membrane were seen through the synaptic basal lamina together with the presynaptic and postsynaptic membranes. This feature of translucency has the potential to analyze possible spatial interrelations in situ between bioactive molecules in the synaptic basal lamina and the synaptic vesicles in further studies. The synaptic basal lamina, appearing as an electron-dense line sandwiched by two parallel lines representing the presynaptic and postsynaptic membranes in ultrathin sections cut right to the synaptic junctional plane in conventional TEM, was not fully continuous but randomly intermittent along its trajectory. Compatible with the intermittent line appearance, the en-face 3D view in embedment-free section TEM revealed for the first time partial irregular defects of the synaptic basal lamina. Considering the known functional significance of several molecules contained in the synaptic basal lamina in the maintenance and exertion of the synapse, its partial defects may not represent its rigid structural features, but its immature structure under remodeling or its dynamic changes in consistency such as the sol/gel transition, whose validity needs further examination. RESEARCH HIGHLIGHTS: In embedment-free section TEM, a 3D en-face view of synaptic basal lamina in situ is reliably possible. The basal lamina en-face is electron-translucent, which makes it possible to analyze spatial interrelation between pre- and post-synaptic components. Partial irregular defects in the basal lamina are revealed in Torpedo electrocytes, suggesting its remodeling or dynamic changes in consistency.

Localization of Diacylglycerol Kinase ζ in the Kidney of Adult Rats: Its Dominant Expression in Collecting Tubules and its Selective Lack in Proximal Tubules / Localización de Diacilglicerol Quinasa ζ en el Riñón de Ratas Adultas: su Expresión Dominante en Túbulos Colectores y su Carencia Selectiva en Túbulos Proximales

SUMMARY: Diacylglycerol kinase (DGK) exerts balancing the intracellular level between two-second messengers, diacylglycerol and phosphatidic acid, by its phosphorylation activity. DGK ζ is often localized in cell nuclei, suggesting its involvement in the regulation of intranuclear activities, including mitosis and apoptosis. The present immunohistochemical study of rat kidneys first revealed no detection levels of DGK ζ -immunoreactivity in nuclei of most proximal tubule epithelia in contrast to its distinct occurrence in cell nuclei of collecting and distal tubules with the former more dominant. This finding suggests that DGK ζ is a key factor regulating vulnerability to acute kidney injury in various renal tubules: its low expression represents the high vulnerability of proximal tubule cells, and its distinct expression does the resistance of collecting and distal tubule cells. In addition, this isozyme was more or less localized in nuclei of cells forming glomeruli as well as in endothelial nuclei of peritubular capillaries and other intrarenal blood vessels, and epithelial nuclei of glomerular capsules (Bowman's capsules) and renal calyces, including intrarenal interstitial cells.

La diacilglicerol quinasa (DGK) ejerce el equilibrio del nivel intracelular entre dos segundos mensajeros, diacilglicerol y ácido fosfatídico, por su actividad de fosforilación. La DGK ζ a menudo se localiza en los núcleos celulares, lo que sugiere su participación en la regulación de las actividades intranucleares, incluidas la mitosis y la apoptosis. El presente estudio inmunohistoquímico en riñones de rata no reveló niveles de detección de inmunorreactividad de DGK ζ en los núcleos de la mayoría de los epitelios de los túbulos proximales, en contraste a la detección en los núcleos celulares de los túbulos colectores y distales, siendo el primero más dominante. Este hallazgo sugiere que DGK ζ es un factor clave que regula la vulnerabilidad a la lesión renal aguda en varios túbulos renales: su baja expresión representa la alta vulnerabilidad de las células del túbulo proximal, y su expresión distinta hace a la resistencia de las células del túbulo colector y distal. Además, esta isoenzima estaba más o menos localizada en los núcleos de las células que forman los glomérulos, así como en los núcleos endoteliales de los capilares peritubulares y otros vasos sanguíneos intrarrenales, y en los núcleos epiteliales de las cápsulas glomerulares (cápsulas de Bowman) y los cálices renales, incluidas las células intersticiales intrarrenales.

Localization of phospholipid-related signal molecules in salivary glands of rodents: A review.

BACKGROUND: In the 1950s, Hokin conducted initial studies on phosphoinositide turnover/cycle in salivary glandular cells. From these studies, the idea emerged that receptor-mediated changes in intramembranous levels of phosphoinositides represent an early step in the stimulus-response pathway. Based on this idea and the general view that knowledge of the exact localization of a given endogenous molecule in cells in situ is important for understanding its functional significance, we have reviewed available information about the localization of several representative phosphoinositide-signaling molecules in the salivary glands in situ in mice. HIGHLIGHT: We focused on phosphatidylinositol 4-kinase, phosphatidylinositol 4 phosphate 5-kinase α, ß, γ, phospholipase Cß, muscarinic cholinoceptors 1 and 3, diacylglycerol kinase ζ, phospholipase D1 and 2, ADP-ribosylation factor 6 and its exchange factors for Arf6, and cannabinoid receptors. These molecules individually exhibit differential localization in a spatiotemporal manner in the exocrine glands, making it possible to deduce their functional significance, such as their involvement in secretion and cell differentiation. CONCLUSION: Although phosphoinositide-signaling molecules whose in situ localization in glandular cells has been clarified are still limited, the obtained information on their localization suggests that their functional significance is more valuable in glandular ducts than in acini. It thus suggests the necessity of greater attention to the ducts in their physio-pharmacological analyses. The purpose of this review is to encourage more in situ localization studies of phosphoinositide-signaling molecules with an aim to further understand their possible involvement in the pathogenesis of salivary gland diseases.

Discrete localization patterns of PIP5Kγ and PLCß3 working sequentially in phosphoinositide-cycle within mouse sensory neuron somata.

It is known that phosphatidylinositol phosphate 5 kinase (PIP5K) γ and phospholipase C (PLC) ß3, 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 PLCß3 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 PLCß3 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, PLCß3-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 PLCß3, but immunopositive for PIP5Kγ. The involvement of PLCß3 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, PLCß3-target, is localized homogenously over the plane of the neuronal plasma membranes. RESEARCH HIGHLIGHTS: PIP5Kγ, different from PLCß3, was localized heterogeneously on neuronal membranes, and this difference was demonstrated in 3D-bright field immuno-light and electron microscopy. Either PIP5Kγ or PLCß3 was not detected in peripheral nerve terminals.

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.

Discrete localization of phospholipase Cß3 and diacylglycerol kinase ι along the renal proximal tubules of normal rat kidney and gentamicin-induced changes in their expression.

Many signaling enzymes have multiple isozymes that are localized discretely at varying molecular levels in different compartments of cells where they play specific roles. In this study, among the various isozymes of phospholipase C (PLC) and diacylglycerol kinase (DGK), which work sequentially in the phosphoinositide cycle, both PLCß3 and DGKι were found in renal brush-border microvilli, but found to replace each other along the proximal tubules: PLCß3 in the proximal straight tubules (PST) of the outer stripe of the outer medulla (OSOM) and the medullary ray (MR), and DGKι in the proximal convoluted tubules (PCT) in the cortex and partially in the PST of the MR. Following daily injection of gentamicin for 1 week, the expression of PLCß3 and DGKι was transiently enhanced, as demonstrated by western blot, and the increases were found to most likely occur in their original sites, that is, in the brush borders of the PST for PLCß3 and in the PCT for DGKι. These findings showing differences in expression along the tubules suggest that the exertion of reabsorption and secretion through various ion channels and transporters in the microvillus membranes and the maintenance of microvillus turnover are regulated by a PLC-mediated signal with the balance shifted toward relative augmentation of the DAG function in the PST, and by a DGK-mediated signal with the balance shifted to relative augmentation of the phosphatidic acid function in the PCT. Our results also suggest the possibility that these isozymes are potential diagnostic signs for the early detection of acute kidney injury caused by gentamicin.

Localization of phosphatidylinositol phosphate 5 kinase γ, phospholipase ß3 and diacylglycerol kinase ζ in corneal epithelium in comparison with conjunctival epithelium of mice.

Based on the theory that the phosphoinositide (PI) signal is involved in the physiology of cornea and conjunctiva, we examined the localization in the mouse anterior ocular epithelia of immunoreactivities for phosphatidylinositol 4-phosphate 5-kinase (PIP5K), phospholipase C (PLC) and diacylglycerol kinase (DGK), enzymes that work sequentially in PI cycle. Immunoreactivity for PIP5Kγ in the corneal epithelium, including the limbus, was distinct in adults in contrast to faint or negligible immunoreactivity in the conjunctival epithelium in neonatal mice. This adult localization pattern was first recognized at the postnatal time of eyelid opening. Immunoreactivity for PLCß3 was rather equally distinct throughout the entire corneal and conjunctival epithelia in adults. DGKζ-immunoreactive nuclei were mainly localized in the basal half domain of the corneal epithelium but in both basal and apical domains of the conjunctival epithelium in adults. This nuclear immunoreactivity was at weak or negligible levels in the peripheral and limbus cornea and in a considerable portion of the bulbar conjunctival epithelium continuous with the limbus. The adult patterns for PLCß3 and DGKζ were already present at birth. The present findings suggest the following possibilities on the functional significance of the three enzyme molecules. PIP5Kγ is involved in cornea-specific functions such as bright-field vision, including corneal transparency, and in the stability of epithelial junctions, for which there seems to be a much higher requirement in the corneal epithelium than in the conjunctival epithelium. PLCß3 is involved from birth in as-yet undefined functions exerted ubiquitously from birth in both corneal and conjunctival epithelia. DGKζ is involved in regulation from birth of the transcription in epithelial cells, including apoptosis as well as regulation of mitosis of epithelial cells in both cornea and conjunctiva, with the transcription involvement more apparent in the conjunctiva, although it does not work in stem cells of the corneal limbus.

Increased Sulfation in Gracilaria fisheri Sulfated Galactans Enhances Antioxidant and Antiurolithiatic Activities and Protects HK-2 Cell Death Induced by Sodium Oxalate.

Urolithiasis is a common urological disease characterized by the presence of a stone anywhere along the urinary tract. The major component of such stones is calcium oxalate, and reactive oxygen species act as an essential mediator of calcium oxalate crystallization. Previous studies have demonstrated the antioxidant and antiurolithiatic activities of sulfated polysaccharides. In this study, native sulfated galactans (N-SGs) with a molecular weight of 217.4 kDa from Gracilaria fisheri were modified to obtain lower molecular weight SG (L-SG) and also subjected to sulfation SG (S-SG). The in vitro antioxidant and antiurolithiatic activities of the modified substances and their ability to protect against sodium oxalate-induced renal tubular (HK-2) cell death were investigated. The results revealed that S-SG showed more pronounced antioxidant activities (DPPH and O2- scavenging activities) than those of other compounds. S-SG exhibited the highest antiurolithiatic activity in terms of nucleation and aggregation, as well as crystal morphology and size. Moreover, S-SG showed improved cell survival and increased anti-apoptotic BCL-2 protein in HK-2 cells treated with sodium oxalate. Our findings highlight the potential application of S-SG in the functional food and pharmaceutical industries.

Mitochondrial Localization of CB1 in Progesterone-producing Cells of Ovarian Interstitial Glands of Adult Mice.

Localization of cannabinoid receptor type 1 (CB1) immunoreactivity on mitochondrial membranes, at least their outer membranes distinctly, was detected in progesterone-producing cells characterized by mitochondria having tubular cristae and aggregations of lipid droplets in ovarian interstitial glands in situ of adult mice. Both immunoreactive and immunonegative mitochondria were contained in one and the same cell. Considering that the synthesis of progesterone is processed in mitochondria, the mitochondrial localization of CB1 in the interstitial gland cells suggests the possibility that endocannabinoids modulate the synthetic process of progesterone in the cells through CB1.

Localization of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) α confined to the surface of lipid droplets and adjacent narrow cytoplasm in progesterone-producing cells of in situ ovaries of adult mice.

Phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) produced by phosphatidylinositol phosphate 5 kinase (PIP5K) plays not only as a precursor of second messengers in the phosphoinositide signal transduction, but also multiple roles influencing a variety of cellular activities. From this viewpoint, the present study attempted to localize PIP5Kα in the ovaries in situ of adult mice. PIP5Kα-immunoreactivity was confined to the surfaces of lipid droplets (LDs) and their adjacent cytoplasm in progesterone-producing cells of the interstitial glands, corpora lutea and theca interna. The LDs often contained membranous tubules/lamellae along their surfaces and within their interior whose membranes were continuous with those delineating LDs composed of a monolayer of phospholipids and were partially PIP5Kα-immunoreactive. Although granulosa cells of healthy-looking follicles were immunonegative, as the atresia progressed, PIP5Kα-immunoreactivity first appeared in sparsely dispersed dot forms in mural cells of the follicular epithelia, and then were dominant in almost all mural cells that remained after desquamation of the antral cells. The present study provides evidence suggesting that PI(4,5)P2 locally synthesized by PIP5K in LDs is involved in the lipid transfer between lipid droplets (LDs) and the endoplasmic reticulum, which eventually regulates ovarian progesterone production through control of multiple dynamic activities of LDs. It is also suggested that PIP5Kα and PI(4,5)P2 are implicated in the modulation of programmed cell death and/or acquiring the ability of progesterone production in some follicular cells surviving atresia.

Spatiotemporally differential expression of HER (human epithelial growth factor receptor)-2 with sexual dimorphism in submandibular gland of mice

SUMMARY: Considering that the submandibular gland (SMG) of postnatal mice performs active cell proliferation, apoptosis and differentiation which are regulated by proto-oncogene products in cancerous cells, the expression and localization of a proto-oncogene product HER (human epidermal growth factor receptor)-2 was examined in SMG of postnatal mice. In Western blot analysis, the expression for HER-2 was high until pre-puberty, and it decreased from puberty to young adult stages with male SMG more dominant. In immunohistochemistry, the immunoreactivity was positive in acinar and ductal cells of newborn SMG with distinct localization at the intercellular apposition sites. The immunoreactivity in acinar cells progressively decreased to negligible levels by pre-pubertal stage, while it remained positive in most ductal cells throughout the postnatal time-course. The immunoreactivity in cells of terminal tubules and intercalated ducts, both of which have a high potential to produce cells, were seen at levels similar to those of more proximal ducts, while the immunoreactivity in ductal basal cells was significantly high, but the granular convoluted tubule cells were seen at negligible levels in male and at faint levels in female. In immuno-electron microscopy of excretory ducts, the immunoreactivity was dominantly localized on the basal infolding membranes as well as vesicles and vacuoles of various sizes, but rarely in Golgi apparatus and mitochondria. The immunoreactivity without association to any membranous structures were also seen, though not numerous. The relation of expression levels of HER-2 in various portions of normal SMG to those in their cancerous ones is briefly discussed.

RESUMEN: Considerando que la glándula submandibular (GSM) de ratones postnatales realiza la proliferación celular activa, apoptosis y diferenciación que están reguladas por productos protooncogénicos en células cancerosas, la expresión y localización de un producto protooncogénico HER (receptor del factor de crecimiento epidérmico humano) - 2 se examinó en GSM de estos ratones. En el análisis de Western blot, la expresión de HER-2 fue alta hasta la prepubertad, y disminuyó desde la pubertad hasta las etapas de adultos jóvenes con GSM macho más dominante. En inmunohistoquímica, la inmunorreactividad fue positiva en las células acinares y ductales de GSM de recién nacido con una localización distinta en los sitios de aposición intercelular. La inmunorreactividad en las células acinares disminuyó progresivamente a niveles insignificantes en la etapa prepuberal, mientras que permaneció positiva en la mayoría de las células ductales durante el transcurso del tiempo posnatal. La inmunorreactividad en las células de los túbulos terminales y los conductos intercalados, los cuales tienen un alto potencial para producir células, se obser- vó a niveles similares a los de los conductos más proximales, mientras que la inmunorreactividad en las células basales ductales fue significativamente alta, pero en el túbulo contorneado granular las células se observaron en niveles insignificantes en los machos y en niveles débiles en las hembras. En la microscopía inmunoelectrónica de los conductos excretores, la inmunorreactividad se localizó de manera predominante en las membranas de pliegues basales, así como en vesículas y vacuolas de varios tamaños, pero raramente en el aparato de Golgi y en las mitocondrias. También se observó la inmunorreactividad sin asociación a ninguna estructura membranosa, aunque no numerosa. Se discute brevemente la relación de los niveles de expresión de HER-2 en varias porciones de GSM normal con aquellos en sus cancerosos.

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.

Increased expression of apoptotic markers in human full-term placenta after exposure to elevated environmental cadmium levels during pregnancy.

Cadmium (Cd), a toxic heavy metal, produces various forms of environmental contaminations and health problems in human. In this study, we aimed to examine the localization of several apoptotic markers in human placentas from pregnant women who were environmentally exposed to Cd. Twelve pregnant women participated in this analysis and they were divided into 2 groups according to their living areas: high-Cd (H-Cd) and low-Cd (L-Cd) groups. After delivery, the placentas were immediately harvested, and the placental width, length, and weight were measured. The placental Cd concentration was determined by using ICP-MS. The expression of three apoptotic markers, cleaved caspase-3, cleaved lamin A/C, and TUNEL, was examined in immunohistochemistry. In results, the placental Cd concentration in the H-Cd group was higher than that in the L-Cd group. In contrast, a significant decrease in the BW (birth weight):PW (placenta weight) ratio representing the placental nutrient transport function was found in the H-Cd group, and an inverse correlation between placental Cd concentration and BW:PW ratio was demonstrated. Additionally, significant elevations in the expression of cleaved caspase-3, cleaved lamin A/C proteins, and TUNEL were shown in the H-Cd placenta. Moreover, positive correlations were found between the placental Cd concentration and the expression of cleaved caspase-3 and TUNEL. Collectively, our findings suggest that the exposure of pregnant women to environmental Cd might induce Cd to be transferred to the body and then accumulated in the placenta, resulting in disturbance of the placental function and eventual apoptosis.

In situ localization of diacylglycerol lipase α and ß producing an endocannabinoid 2-arachidonoylglycerol and of cannabinoid receptor 1 in the primary oocytes of postnatal mice.

In order to understand the mechanism of the endocannabinoid (eCB) signal, which has so far been shown to work in oocyte genesis and maturation, it is critical to clarify detailed localization of the eCB synthesizing enzyme molecules as well as receptors for eCBs in oocytes in the ovary in situ. For this purpose, diacylglycerol lipase (DGL) α and ß are involved in the synthesis of an eCB 2-arachidonoylglycerol (2-AG). DGLα/ß and the cannabinoid receptor 1 (CB1) for 2-AG were shown to be localized to the primary oocytes of postnatal mice using immuno-light and electron microscopy. It was found that two types of localization existed: first, immunoreactivities for DGLα and ß were weakly detected throughout the ooplasm in light microscopy for which the intracellular membranes of vesicles forming tiny scattered aggregates were responsible. Secondly, DGLß-immunoreactivity was distinctly confined to the nuage of Balbiani bodies and small nuage-derivative structures; both amorphous materials and membranes of vesicles were responsible for their localization. On the other hand, the weak immunoreactivity for CB1 was localized in a pattern similar to the first one for DGLs, but not found in a pattern for the Balbiani nuage. Two routes of functional exertion of 2-AG synthesized by DGLs were suggested from the two types of localization: one was that the eCB synthesized at all the sites of DGLs is released from the oocytes and exerts paracrine or autocrine effects on adjacent intra-ovarian cells as well as the oocytes themselves. The other was that the eCB synthesized within the nuage was involved in the modulation of the posttranscriptional processing of oocytes. Owing to the failure in the detection of CB1 in the Balbiani nuage, however, the validity of the latter possibility remains to be elucidated.

Different expression and subcellular localization of vesicular inhibitory amino acid transporter in ducts of major salivary glands: An in situ study in mice.

OBJECTIVE: The aim of this study was to clarify the mechanism of GABA (□-amino butyric acid)-signaling in the salivary glands by localization of vesicular inhibitory amino acid transporter, a key molecule in GABA-synthesis. DESIGN: Parotid, sublingual and submandibular glands of mice at various postnatal stages were examined in immuno-light and electron microscopy as well as immuno-blotting. RESULTS: Expression for vesicular inhibitory amino acid transporter was detected in parotid and sublingual glands of both sexes and female submandibular gland throughout postnatal development, while it was negligible in male submandibular glands at and after puberty. The expression in female submandibular glands attenuated after testosterone injection. The immunoreactivity was localized in striated ductal cells, but not acinar cells, in the salivary glands, and it occurred in association with intracellular and plasma membranes of the cells. It also occurred in myoepithelial and vascular smooth muscle cells. CONCLUSIONS: GABA-signaling was suggested to be a significant signaling pathway in salivary ductal cells, which was suppressed in male submandibular glands at and after puberty. The suppression in the submandibular duct was by testosterone. In addition, the participation of vesicular inhibitory amino acid transporter in GABA signaling through plasma membranes of the ductal cells was suggested. The significance of occurrence of the immunoreactivity in myoepithelial and smooth muscle cells remains to be further elucidated in terms of implication in GABA signaling.

Discrete localization patterns of Arf6, and its activators EFA6A and BRAG2, and its effector PIP5kinaseγ on myofibrils of myotubes and plasma membranes of myoblasts in developing skeletal muscles of mice.

Arf6 (ADP ribosylation factor 6), activated by Arf-GEF (guanine nucleoside exchange factor), is involved in the membrane trafficking and actin-remodeling which are critical for maintenance of cell organization and activity and for fusion of myoblasts to form myotubes/myofibers. EFA6A (exchange factor for Arf6 type A) and BRAG2 (brefeldin A-resistant Arf-GEF 2) represent members of discrete subfamilies of Arf-GEF, while PIP5Kγ (phosphatidylinositol4-phosphate5-kinase γ) produces PI 4,5-bisphosphate (PIP2) and it is target for Arf6. In the present study, immunoreactive bands for Arf6, EFA6A, BRAG2 and PIP5Kγ were detected in immunoblots of skeletal muscle homogenates of mice at E18D (embryonic day 18), while the bands for Arf6, EFA6A and PIP5Kγ were reduced in density and no significant bands for BRAG2 were discerned at P1D (postnatal 1 day). No immunoblot bands for any of the molecules were eventually detected in skeletal fibers of adult mice. Immunoreactivities for endogenous Arf6, EFA6A and PIP5Kγ were visualized using immuno-light microscopy localized as periodic striations running perpendicular to the longitudinal axes of skeletal muscle fibers of mice at E18D and P1D. All the striations were co-immunoreactive for ß-actin in double immunofluorescence microscopy, and the immunoreactivities were confined to thin myofilaments at sarcomeric I-domains in immuno-electron microscopy. On the other hand, immunoreactivities for Arf6, BRAG2 and PIP5Kγ were conspicuous on plasmalemma of myoblasts at E14D, while immunoreactivity for EFA6A was already distinct in striations perpendicular to myofibrils in myotubes at E14D. The present findings suggest three possibilities: involvement of EFA6A-activated Arf6 together with PIP5Kγ in maturation of myofibrils, movement of Arf6 and PIP5Kγ from the plasmalemma of myoblasts to myofibrils of myotubes, and that of BRAG2 to the cytoplasm of myotubes; and further a function of EFA6A independent of the activation of Arf6 in immature myofibrils. In addition, the involvement of Arf6, BRAG2 and PIP5Kγ in the fusion of myoblasts into myotubes was supported by the present finding.

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.

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 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.