The existence of cells exhibiting characteristics of both Type II and Type III cells in rat taste buds. An immunohistochemical and electron-microscopic study.

Taste bud cells are classified into four types by their ultrastructural features. Immunohistochemical detection of taste-signaling molecules is used to distinguish cell types of taste bud cells; however, the characteristics of taste cell types such as the immunoreactivity for taste-signaling molecules have long remained unclear. We investigated the detailed characteristics of taste cells in rat vallate taste buds by electron microscopy and immunohistochemistry for gustducin, neural cell adhesion molecule (NCAM) and vesicle-associated membrane protein 2 (VAMP2), which are known as markers of Type II cells, Type III cells and both cell types, respectively. Triple immunostaining for these molecules discriminated seven kinds of cell, including the totally immunopositive cell. Electron microscopy revealed Type III cells with a typical synaptic structure and subsurface cisterna as a specialized contact between a nerve and a Type II cell. The present study clarified the existence of cells with features of both Type II and Type III cells as a subtype of taste bud cells in the rat taste bud.

The small GTPase Rab33A participates in regulation of amylase release from parotid acinar cells.

Amylase is released from exocrine parotid acinar cells via typical exocytosis. Exocytosis of amylase-containing granules occurs through several steps, including formation, maturation, and transport of granules. These steps are thought to be regulated by members of the small GTPase Rab family. We previously demonstrated that Rab27 and its effectors mediate amylase release from parotid acinar cells, but the functional involvement of other Rab proteins in exocrine granule exocytosis remains largely unknown. Here, we studied isoproterenol (IPR)-induced amylase release from parotid acinar cells to investigate the possible involvement of Rab33A, which was recently suggested to regulate exocytosis in hippocampal neurons and PC12 cells. Rab33A was endogenously expressed in parotid acinar cells and present in secretory granules and the Golgi body. Functional ablation of Rab33A with anti-Rab33A antibody or a dominant-negative Rab33A-T50N mutant significantly reduced IPR-induced amylase release. Our results indicated that Rab33A is a novel component of IPR-stimulated amylase secretion from parotid acinar cells.

Dynamics of testis-ova in a wild population of Japanese pond frogs, Rana nigromaculata.

Although many studies have reported the occurrence of testis-ova in wild frog populations, the origin and trigger of testis-ova differentiation/development remain unclear. A high frequency of testis-ova has been previously reported for wild populations of the Japanese pond frog, Rana nigromaculata (cf. Iwasawa and Asai, '59). In the present study, we aimed to clarify the dynamics of testis-ova in this frog species, including the origin and artificial induction of testis-ova. Testis-ova were observed in both mature frogs and puberty-stage frogs (i.e., 0- and 1-year-old frogs). However, the early stages of testis-ova (~pachytene stage) were mostly observed in puberty-stage male frogs at the onset of spermatogenesis. The early stages of testis-ova were observed in the cysts of early secondary spermatogonia and the single cysts of the primary spermatogonium. This finding indicates that testis-ova differentiation occurs during spermatogonial proliferation and that it is correlated with the initiation of spermatogenesis. We also examined whether estrogen exposure induced testis-ova differentiation and how it is correlated with the progression of spermatogenesis. When 1-year-old frogs were exposed to estradiol-17ß during spring (i.e., when spermatogenesis was initiated), testis-ova differentiation was induced in a dose-dependent manner. However, this phenomenon did not occur in 1-year-old frogs during summer, (i.e., when the transition from spermatogonia to spermatocytes mainly occurs). These results present the first evidence that testis-ova of the Japanese pond frog are derived from primary and early secondary spermatogonia, and that estrogen exposure induces testis-ova differentiation accompanied by the initiation of spermatogenesis.

Characteristics of alveolar bone associated with physiological movement of molar in mice: a histological and histochemical study.

Mouse molars undergo distal movement, during which new bone is formed at the mesial side of the tooth root whereas the preexisting bone is resorbed at the distal side of the root. However, there is little detailed information available regarding which of the bones that surround the tooth root are involved in physiological tooth movement. In the present study, we therefore aimed to investigate the precise morphological differences of the alveolar bone between the bone formation side of the tooth root, using routine histological procedures including silver impregnation, as well as by immunohistochemical analysis of alkaline phosphatase and tartrate-resistant acid phosphatase activity, and immunohistochemical analysis of the expression of the osteocyte markers dentin matrix protein 1, sclerostin, and fibroblast growth factor 23. Histochemical analysis indicated that bone formation by osteoblasts and bone resorption by osteoclasts occurred at the bone formation side and the bone resorption side, respectively. Osteocyte marker immunoreactivity of osteocytes at the surface of the bone close to the periodontal ligament differed at the bone formation and bone resorption sides. We also showed different specific features of osteocytic lacunar canalicular systems at the bone formation and bone resorption sides by using silver staining. This study suggests that the alveolar bone is different in the osteocyte nature between the bone formation side and the bone resorption side due to physiological distal movement of the mouse molar.

Atrophy of myoepithelial cells in parotid glands of diabetic mice; detection using skeletal muscle actin, a novel marker.

In mouse parotid glands, we found expression of skeletal muscle actin (actin-α1) protein and mRNA. We isolated myoepithelial cells from the mouse parotid glands and investigated their actin-α1 expression because smooth muscle actin (actin-α2) has been used as a marker for myoepithelial cells. We used actin-α1 expression to identify pathological changes in diabetic non-obese diabetic (NOD; NOD/ShiJcl) mice-a mouse model for Sjögren's syndrome-and found myoepithelial cells to be decreased or atrophied in the diabetic state.

Occurrence of gustducin-immunoreactive cells in von Ebner's glands of guinea pigs.

An immunohistochemical examination of guinea-pig taste buds in vallate papillae revealed gustducin-immunoreactive cells in the area of von Ebner's glands, minor salivary glands. Since there have been no reports describing those cells in these locations for other species, we investigated these glands in order both to localize the cells and compare their immunoreactive characteristics with corresponding cells in the vallate taste buds. The gustducin-immunoreactive cells coincided with cells containing no secretory granules in the end portion of the glands, which was supported by the electron-microscopic immunocytochemistry. Double immunofluorescence microscopy confirmed these cells to be entirely immunopositive to type III inositol 1,4,5-triphosphate receptor (IP3R-3), phospholipase Cß2 (PLCß2), and villin and also partly immunopositive to neuron-specific enolase (NSE) and calbindin D-28K. The gustducin-immunoreactive cells in the vallate taste buds exhibited completely the same immunoreactivities for these five molecules. Accordingly, the present results give credence to a consideration that the gustducin-immunnoreactive cells in both locations are identical in function(s) e.g., chemo-reception.

Antigen-presenting cells in parotid glands contain cystatin D originating from acinar cells.

Cystatin D encoded by Cst5 is a salivary classified type II cystatin. We investigated the dynamism of cystatin D by examining the distribution of cystatin D protein and mRNA in rats, to identify novel functions. The simultaneous expression of Cst5 and cystatin D was observed in parotid glands, however in situ hybridization showed that only acinar cells produced cystatin D. Synthesized cystatin D was localized in small vesicles and secreted from the apical side to the saliva, and from the basolateral side to the extracellular region, a second secretory pathway for cystatin D. We also identified antigen-presenting cells in the parotid glands that contained cystatin D without the expression of Cst5, indicating the uptake of cystatin D from the extracellular region. Cystatin D was detected in blood serum and renal tubular cells with megalin, indicating the circulation of cystatin D through the body and uptake by renal tubular cells. Thus, the novel dynamism of cystatin D was shown and a function for cystatin D in the regulation of antigen-presenting cell activity was proposed.

The epithelial sodium channel in the Australian lungfish, Neoceratodus forsteri (Osteichthyes: Dipnoi).

Epithelial sodium channel (ENaC) is a Na(+)-selective, aldosterone-stimulated ion channel involved in sodium transport homeostasis. ENaC is rate-limiting for Na(+) absorption in the epithelia of osmoregulatory organs of tetrapods. Although the ENaC/degenerin gene family is proposed to be present in metazoans, no orthologues or paralogues for ENaC have been found in the genome databases of teleosts. We studied full-length cDNA cloning and tissue distributions of ENaCα, ß and γ subunits in the Australian lungfish, Neoceratodus forsteri, which is the closest living relative of tetrapods. Neoceratodus ENaC (nENaC) comprised three subunits: nENaCα, ß and γ proteins. The nENaCα, ß and γ subunits are closely related to amphibian ENaCα, ß and γ subunits, respectively. Three ENaC subunit mRNAs were highly expressed in the gills, kidney and rectum. Amiloride-sensitive sodium current was recorded from Xenopus oocytes injected with the nENaCαßγ subunit complementary RNAs under a two-electrode voltage clamp. nENaCα immunoreactivity was observed in the apical cell membrane of the gills, kidney and rectum. Thus, nENaC may play a role in regulating sodium transport of the lungfish, which has a renin-angiotensin-aldosterone system. This is interesting because there may have been an ENaC sodium absorption system controlled by aldosterone before the conquest of land by vertebrates.

Exocyst subunits are involved in isoproterenol-induced amylase release from rat parotid acinar cells.

Exocytosis of secretory granules in parotid acinar cells requires multiple events: tethering, docking, priming, and fusion with a luminal plasma membrane. The exocyst complex, which is composed of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) that are conserved in yeast and mammalian cells, is thought to participate in the exocytotic pathway. However, to date, no exocyst subunit has been identified in salivary glands. In the present study, we investigated the expression and function of exocyst subunits in rat parotid acinar cells. The expression of mRNA for all eight exocyst subunits was detected in parotid acinar cells by RT-PCR, and Sec6 and Sec8 proteins were localized on the luminal plasma membrane. Sec6 interacted with Sec8 after 5 min of stimulation with isoproterenol. In addition, antibodies to-Sec6 and Sec8 inhibited isoproterenol-induced amylase release from streptolysin O-permeabilized parotid acinar cells. These results suggest that an exocyst complex of eight subunits is required for amylase release from parotid acinar cells.

EPI64 protein functions as a physiological GTPase-activating protein for Rab27 protein and regulates amylase release in rat parotid acinar cells.

Rab27, a small GTPase, is generally recognized as an important regulator of secretion that interacts with Rab27-specific effectors to regulate events in a wide variety of cells, including endocrine and exocrine cells. However, the mechanisms governing the spatio-temporal regulation of GTPase activity of Rab27 are not firmly established, and no GTPase-activating protein (GAP) specific for Rab27 has been identified in secretory cells. We previously showed that expression of EPI64, a Tre-2/Bub2/Cdc16 (TBC)-domain-containing protein, in melanocytes inactivates endogenous Rab27A on melanosomes (Itoh, T., and Fukuda, M. (2006) J. Biol. Chem. 281, 31823-31831), but the EPI64 role in secretory cells has never been investigated. In this study, we investigated the effect of EPI64 on Rab27 in isoproterenol (IPR)-stimulated amylase release from rat parotid acinar cells. Subcellular fractionation and immunohistochemical analyses indicated that EPI64 was enriched on the apical plasma membrane of parotid acinar cells. We found that an antibody against the TBC/Rab-GAP domain of EPI64 inhibited the reduction in levels of the endogenous GTP-Rab27 in streptolysin-O-permeabilized parotid acinar cells and suppressed amylase release in a dose-dependent manner. We also found that the levels of EPI64 mRNA and EPI64 protein increased after IPR stimulation, and that treatment with actinomycin D or antisense-EPI64 oligonucleotides suppressed the increase of EPI64 mRNA/EPI64 protein and the amount of amylase released. Our findings indicated that EPI64 acted as a physiological Rab27-GAP that enhanced GTPase activity of Rab27 in response to IPR stimulation, and that this activity is required for IPR-induced amylase release.

A morphological analysis on the osteocytic lacunar canalicular system in bone surrounding dental implants.

Osseointegration is the most preferable interface of dental implants and newly formed bone. However, the cavity preparation for dental implants often gives rise to empty lacunae or pyknotic osteocytes in bone surrounding the dental implant. This study aimed to examine the chronological alternation of osteocytes in the bone surrounding the titanium implants using a rat model. The distribution of the osteocytic lacunar canalicular system (OLCS) in bone around the titanium implants was examined by silver impregnation according to Bodian's staining. We also performed double staining for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), as well as immunohistochemistry for fibroblast growth factor (FGF) 23--a regulator for the serum concentration of phosphorus--and sclerostin, which has been shown to inhibit osteoblastic activities. Newly formed bone and the injured bone at the early stage exhibited an irregularly distributed OLCS and a few osteocytes positive for sclerostin or FGF23, therefore indicating immature bone. Osteocytes in the surrounding bone from Day 20 to Month 2 came to reveal an intense immunoreactivity for sclerostin. Later on, the physiological bone remodeling gradually replaced such immature bone into a compact profile bearing a regularly arranged OLCS. As the bone was remodeled, FGF23 immunoreactivity became more intense, but sclerostin became less so in the well-arranged OLCS. In summary, it seems likely that OLCS in the bone surrounding the dental implants is damaged by cavity formation, but later gradually recovers as bone remodeling takes place, ultimately inducing mature bone.

Fixation conditions affect the immunoreactivity of gustducin in rat vallate taste buds.

This study aimed to examine gustducin immunoreactivities when adopting various immunostaining conditions in rat vallate taste buds. The occurrence and intensity of the immunoreactivities exhibited specific patterns in accordance with the fixation time. The immunoreactions were localized to only taste hairs, the upper part of the taste bud, after short fixation periods but then to the cell-body cytoplasm excluding the taste hairs after long fixation periods. These immunohistochemical data suggest that the staining protocols, especially the fixation time, cause discrepancies in gustducin immunoreactivities.

Redistribution of small GTP-binding protein, Rab27B, in rat parotid acinar cells after stimulation with isoproterenol.

Small GTP-binding protein, Rab27, has been implicated in the regulation of different types of membrane trafficking, including melanosome transport in melanocytes and regulated secretion events in a wide variety of secretory cells. We have previously shown that Rab27 is involved in the control of isoproterenol (IPR)-induced amylase release from rat parotid acinar cells. Although Rab27 is predominantly localized on secretory granules under resting conditions, changes to its intracellular localization after beta-stimulation have never been elucidated. The present study investigated IPR-induced redistribution of Rab27B in the parotid acinar cells, revealing translocation from secretory granules to the subapical region after 5 min of IPR treatment and then diffusion into the cytosol after 30 min of IPR treatment. Dissociation of Rab27B from the apical plasma membrane is probably mediated through the Rab GDP dissociation inhibitor (GDI) in the cytosol extracting GDP-bound Rab protein from membranes, as a dramatic increase in the amount of the Rab27B-GDI complex in the cytosol was observed 30 min after stimulation with IPR. These results indicate that, in parotid acinar cells, Rab27B is translocated, in a time-dependent manner, from secretory granules into the apical plasma membrane as a result of exposure to IPR, and then into the cytosol through binding with the GDI.

Transferrin secretory pathways in rat parotid acinar cells.

Transferrin is the major iron transporter in blood plasma, and is also found, at lower concentrations, in saliva. We studied the synthesis and secretion of transferrin in rat parotid acinar cells in order to elucidate its secretory pathways. Two sources were identified for transferrin in parotid acinar cells: synthesis by the cells (endogenous), and absorption from blood plasma (exogenous). Transferrin from both sources is secreted from the apical side of parotid acinar cells. Endogenous transferrin is transported to secretory granules. It is secreted from mature secretory granules upon stimulation with a beta-adrenergic reagent and from smaller vesicles in the absence of stimulation. Exogenous transferrin is internalized from the basolateral side of parotid acinar cells, transported to the apical side by transcytosis, and secreted from the apical side. Secretory processes for exogenous transferrin include transport systems involving microfilaments and microtubules.

Redistribution of Rab27-specific effector Slac2-c, but not Slp4-a, after isoproterenol-stimulation in rat parotid acinar cells.

Small GTPase Rab27 has been implicated in the regulation of different types of membrane trafficking, including melanosome transport and various regulated secretion events. We have previously shown that Rab27 and its effectors, Slac2-c/MyRIP and Slp4-a/granuphilin-a, are involved in the control of isoproterenol (IPR)-induced amylase release from rat parotid acinar cells. The ability of Rab to interact with the specific effectors is important. However, little is known about the fate of these effectors after beta-adrenergic stimulation in parotid acinar cells. The present study investigated changes in intracellular redistribution of Slac2-c and Slp4-a in parotid acinar cells after IPR treatment. Subcellular fractionation studies detected Slac2-c and Slp4-a in the apical plasma membrane (APM) and secretory granules under resting conditions. After 5min of IPR treatment, Slac2-c was rapidly recruited to the luminal site, but after 30 min, the amount of Slac2-c in the APM fraction was reduced by approximately 80% compared to the increased level after 5 min of IPR treatment. Such reductions in Slac2-c are likely caused by the translocation of Slac2-c from the APM to the cytosol. In addition, we found that Slac2-c in the cytosolic fraction, but not other fractions, disappeared in the presence of Ca(2+). Since Slac2-c contains multiple PEST-like sequences (i.e., potential signals for rapid protein degradation), we suggest that Slac2-c is Ca(2+)-dependently proteolyzed in the cytosol after exocytosis. In contrast, intracellular localization and expression levels of Slp4-a in parotid acinar cells were unaltered even after beta-stimulation, indicating completely different fates for the two Rab27 effectors after beta-stimulation.

Receptosecretory nature of type III cells in the taste bud.

Type III cells in taste buds form chemical synapses with intragemmal afferent nerve fibers and are characterized by the presence of membrane-bound vesicles in the cytoplasm. Although the vesicles differ in shape and size among species, they are primarily categorized into small clear (40 nm in diameter) and large dense-cored (90-200 nm) types. As such vesicles tend to be closely juxtaposed to the synaptic membrane of the cells, it is reasonable to consider that the vesicles include transmitter(s) towards the gustatory nerve. In the guinea-pig taste bud, stimulation with various taste substances (sucrose, sodium chloride, quinine hydrochloride, or monosodium L-glutamate) causes ultrastructural alterations of the type III cells. At the synapse, the presynaptic plasma membrane often displays invaginations of 90 nm in a mean diameter towards the cytoplasm, which indicates the dense-cored vesicles opening into the synaptic cleft by means of exocytosis. The vesicles are also exocytosed at the non-synaptic region into the intercellular space. These findings strongly suggest that the transmitters presumably contained in the vesicles are released to conduct the excitement of the type III cells to the nerves and also to exert their paracrine effects upon the surroundings, such as the Ebner's salivary gland, acting as local hormones.

Unstimulated amylase secretion is proteoglycan-dependent in rat parotid acinar cells.

It is well-known that amylase is secreted in response to extracellular stimulation from the acinar cells. However, amylase is also secreted without stimulation. We distinguished vesicular amylase as a newly synthesized amylase from the accumulated amylase in secretory granules by short time pulse and chased with (35)S-amino acid. The newly synthesized amylase was secreted without stimulation from secretory vesicles in rat parotid acinar cells. The secretion process did not include microtubules, but was related to microfilaments. p-Nitrophenyl beta-xyloside, an inhibitor of proteoglycan synthesis, inhibited the newly synthesized amylase secretion. This indicated that the newly synthesized amylase was secreted from secretory vesicles, not via the constitutive-like secretory route, which includes the immature secretory granules, and that proteoglycan synthesis was required for secretory vesicle formation.

mGluR1 in the fundic glands of rat stomach.

l-glutamate not only confers cognitive discrimination for umami taste in the oral cavity, but also conveys sensory information to vagal afferent fibers in the gastric mucosa. We used RT-PCR, western blotting, and immunohistochemistry to demonstrate that mGluR1 is located in glandular stomach. Double staining revealed that mGluR1 is found at the apical membrane of chief cells and possibly in parietal cells. Moreover, a diet with 1% l-glutamate induced changes in the expression of pepsinogen C mRNA in stomach mucosa. These data suggest that mGluR1 is involved in the gastric phase regulation of protein digestion.

Existence of subtypes of gustducin-immunoreactive cells in the vallate taste bud of guinea pigs.

Vallate taste buds in the guinea-pig tongue were immunohistochemically investigated with regard to the colocalization of gustducin with calbindin-D28K (=spot 35 protein) and type III inositol triphosphate receptor (IP(3)R-3) in order to characterize gustducin-immunoreactive cells. Individual taste bud cells ranged from totally immunopositive to totally immunonegative for these three molecules. Among the immunoreactive cells, gustducin-immunoreactive cells were divided into two cell populations: one immunopositive and the other immunonegative for calbindin-D28K. Applying our previous data to the present results, the former cells should belong to Type III cells designated by electron microscopy. This finding provides new evidence regarding the taste bud types of cells expressing gustducin in the guinea pig.

Functional involvement of Noc2, a Rab27 effector, in rat parotid acinar cells.

Noc2 has recently been proposed to regulate exocytosis in both endocrine and exocrine cells; however, protein expression, subcellular localization and function of Noc2 in exocrine cells have never been elucidated. In this study, we investigated whether Noc2, a Rab27 effector, is involved in isoproterenol (IPR)-stimulated amylase release from acinar cells. Rab27 was detected in the apical plasma membrane (APM) and secretory granule membrane (SGM) fractions, and was translocated to the APM after IPR stimulation for 5 min, but was detected at lower levels in the APM after 30 min. In contrast, although Noc2 was expressed in SGM bound to Rab27, Noc2 was not translocated to APM and the Noc2/Rab27 complex was disrupted after stimulation with IPR for short time. In addition, the anti-Noc2-Rab-binding-domain antibody inhibited IPR-stimulated amylase release from streptolysin O-permeabilized parotid acinar cells. Our results suggest that the Noc2/Rab27 complex is an important constituent of the early stages of IPR-stimulated amylase release.