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Takashi Toyono

Publications and source records attributed to Takashi Toyono.

16 recordsLinked to original sources
PubMed2007

Expression of group II metabotropic glutamate receptors in rat gustatory papillae.

Glutamate is one candidate for the neurotransmitters and/or neuromodulators involved in taste signaling in taste buds. Group II metabotropic glutamate receptors (mGluRs: mGluR2 and mGluR3) are known to function as presynaptic receptors that regulate the release of glutamate and/or other neurotransmitters in the central nervous system. Group II mGluRs are negatively linked to adenylyl cyclase through Galphai subunits and thereby reduce the turnover of cAMP. In rat taste tissues, a subset of adenylyl-cyclase-8-expressing taste cells coexpress the Galphai subunits gustducin and Galphai2. However, the expression patterns of group II mGluRs in rat taste tissues have not yet been elucidated. We have therefore examined the expression patterns of mGluR2, mGluR3, and gustducin in rat gustatory tissues. Reverse transcription/polymerase chain reaction assays have revealed that mGluR2 and mGluR3 mRNAs are expressed in the circumvallate papillae. In situ hybridization analyses have detected positive signals for mGluR2 and mGluR3 mRNAs only in the circumvallate taste buds. Among the fungiform, foliate, and circumvallate papillae, an antibody against mGluR2/3 labels a subset of taste bud cells and nerve fibers immediately beneath the taste lingual epithelium. Double-labeling experiments have demonstrated that mGluR2/3-positive cells coexpress gustducin. These results indicate that mGluR2 and mGluR3 are coupled to Galphai subunits and play roles in glutamate-mediated signaling in taste transductions.

Animals↗
PubMed2007

Immunohistochemical localization of aromatic L-amino acid decarboxylase in mouse taste buds and developing taste papillae.

Aromatic L-amino acid decarboxylase (AADC) catalyses the decarboxylation of all aromatic L-amino acids. In mammals, AADC is expressed in many tissues besides the nervous system, and is associated with additional regulatory roles of dopamine and serotonin in a wide range of tissues. We examined the expression of AADC by using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. RT-PCR analysis showed that mRNA of AADC was detected in the taste bud-containing epithelium of the circumvallate papilla of mice. By immunohistochemical analyses, AADC was detected in a subset of taste bud cells of fungiform, foliate, and circumvallate papillae. Double-label studies showed that AADC colocalized with serotonin, NCAM, PLCbeta2, and PGP9.5. On the other hand, AADC never colocalized with alpha-gustducin. Our results of double staining with AADC and taste cell markers indicate that only the type III cells could convert 5-hydroxytryptophan (5-HTP) to serotonin within taste buds. Taken together with previous studies, the properties of the type III cell of taste buds exactly fit into the APUD (amine and amine precursor uptake and decarboxylation) cell scheme. Furthermore, in the developing circumvallate papilla, AADC are first detected in a small number of papillary epithelial cells at E14.5. By E18.5, AADC-positive epithelial cells also express PGP9.5, which is one of marker of taste cells, and these cells have been contacted by developing nerve fibers. These results suggest that AADC expression begins at early stages of taste bud cell differentiation, and biogenic amines may act on taste bud differentiation of tongue epithelial cells, and further may regulate innervation of taste bud progenitor cells.

Animals↗
PubMed2006

The bHLH transcription factors, Hes6 and Mash1, are expressed in distinct subsets of cells within adult mouse taste buds.

Taste buds are multicellular receptor organs embedded in the lingual epithelium of vertebrates. Taste cells within these buds are modified epithelial cells as they lack axons and turnover rapidly throughout life, yet have neuronal properties enabling them to transduce taste stimuli and transmit this information to the nervous system. Taste cells are heterogeneous, comprising types I, II, III and basal cells, and are continually replaced during adult life, raising the question of how these different cells are generated. The molecular mechanisms governing taste cell differentiation are unknown, but the Notch signaling system has been implicated in this process based upon recent gene expression data. Here we investigate the expression in mature taste buds of Notch related transcription factors, Hes6 and Mash1, which are among the first genes expressed in embryonic taste buds. We further compare these patterns with those of immunocytochemical markers of discrete taste cell types. We find that Hes6 is expressed in a subset of basally located, possibly progenitor cells, yet is rarely coexpressed with taste cell markers. In contrast, Mash1 is detected in some basal cells and in the majority of differentiated type III taste cells, but never in type II cells. These data suggest a role for Notch signaling in taste cell differentiation in adult taste buds.

Animals↗
PubMed2005

Transient outward K+ currents in rat dissociated subfornical organ neurones and angiotensin II effects.

Although angiotensin II inhibits transient outward K+ currents (I(A)s) in subfornical organ neurones, there is no evidence concerning which Kv channels are involved. We investigated I(A)-generating Kv channels in dissociated rat subfornical organ neurones, using molecular, electrophysiological and pharmacological techniques, and studied the effects of angiotensin II. Conventional RT-PCR showed the presence of mRNAs for channels of the Kv3.4, Kv1.4 and Kv4 families, which are capable of generating I(A)s. Tetraethylammonium at 1 mm, which blocks Kv3 channel-derived currents, and blood-depressing substance-I, a Kv3.4-specific blocker, at 2 microm suppressed the I(A)-like component of whole-cell outward currents in some neurones. 4-Aminopyridine at 5 mm inhibited I(A)s in the presence of tetraethylammonium at 1 mm. Cd2+ at 300 microm shifted the activation and inactivation curves of the 4-aminopyridine-sensitive and tetraethylammonium-resistant I(A)s positively. The tetraethylammonium-resistant I(A)s showed fast and slow components during the process of recovery from inactivation, but the slow component was not seen in all neurones. The time constant of the fast recovery component was less than 200 ms, while that of the slow recovery component was around 1 s. Using single-cell RT-PCR, mRNAs for Kv4.2 and Kv4.3L were detected frequently, but those for Kv1.4 and Kv3.4 were seen only rarely. Angiotensin II at 30 nm inhibited the fast recovery component of tetraethylammonium-resistant I(A)s in many neurones. These results suggest that the fast recovery component of the tetraethylammonium-resistant I(A) in subfornical organ neurones depends upon Kv4, and that it can be modulated by angiotensin II.

Angiotensin II↗
PubMed2005

In vitro differentiation of dental epithelial progenitor cells through epithelial-mesenchymal interactions.

In developing teeth, dental epithelial progenitor cells differentiate through sequential and reciprocal interactions with neural-crest-derived mesenchyme. However, the molecular mechanisms involved in cell differentiation are not well understood. Continuously growing teeth are useful in the study of differentiation of dental progenitor cells. In rat lower incisors, ameloblasts originate from the dental epithelial adult stem cell compartment referred to as the 'apical bud'. To elucidate the mechanism of ameloblast differentiation, we designed a primary culture system and confirmed the differentiation of dental epithelial cells through interaction with mesenchymal cells. Cytokeratin was used as a marker for epithelial cells, nerve growth factor receptor p75 for inner enamel epithelial (IEE) cells, and ameloblastin for ameloblasts. The apical bud cells could only differentiate into IEE cells and, within 10 days, into ameloblasts expressing ameloblastin in the presence of dental papilla cells. Interestingly, the IEE cells could proliferate transiently and differentiate into ameloblasts in the presence or absence of dental papilla cells. These results suggest that apical bud cells can enter the ameloblast cell lineage through interaction with mesenchymal cells. IEE cells, on the other hand, are already committed to differentiate into ameloblasts. This culture system is useful in future studies of ameloblast differentiation.

Ameloblasts↗
PubMed2005

Expression of synaptotagmin 1 in the taste buds of rat gustatory papillae.

Synapses between taste receptor cells and primary sensory afferent fibers transmit the output signal from taste buds to the central nervous system. The synaptic vesicle cycle at the synapses involves vesicle docking, priming, fusion, endocytosis, and recycling. Many kinds of synaptic vesicle proteins participate in synaptic vesicle cycles. One of these, synaptotagmin 1, binds Ca(2+) phospholipids with high affinity and plays a role in Ca(2+) regulated neurotransmitter release in the central and peripheral nervous systems. However, the expression patterns of synaptotagmin 1 in rat taste tissues have not been determined. We therefore examined the expression patterns of synaptotagmin 1 and several cell specific markers of type II and III cells in rat taste buds. RT-PCR assay showed that synaptotagmin 1 mRNA was expressed in circumvallate papillae. In fungiform, foliate, and circumvallate papillae, the antibody against synaptotagmin 1 yielded the labeling of a subset of taste bud cells and intra- and subgemmal nerve processes. Double labeled experiments showed that synaptotagmin 1 positive cells co-expressed type III cell markers, PGP 9.5, and NCAM. Intragemmal nerve processes positive for synaptotagmin 1 co-expressed PGP 9.5. Conversely, all synaptotagmin 1 expressing cells did not co-expressed type II cell markers, PLCbeta2, or gustducin. These results show that synaptotagmin 1 may play some regulatory roles in vesicle membrane fusion events with the plasma membrane at the synapses of type III cells in rat taste buds.

Animals↗
PubMed2004

Expression of P2Y1 receptors in rat taste buds.

Extracellular nucleotides such as ATP are the signaling molecules which bind to membrane receptors (P2X ligand-gated ion channels and G-protein-coupled P2Y families). In the gustatory system, it is known that P2X receptors are expressed exclusively in nerve fibers innervating the taste buds. Also, P2Y receptors are suggested to play some important roles in taste signal transductions on the basis of the physiological studies. In the present study, we examined the expression patterns of P2Y1 receptor subtype by using reverse transcript polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. RT-PCR analyses showed that P2Y1 receptor mRNAs appeared in circumvallate papillae. P2Y1 receptor mRNA was detected in a subset of taste bud cells by in situ hybridization. By immunohistochemical analyses, P2Y1 receptor was detected in a subset of taste bud cells of fungiform, foliate, and circumvallate papillae. We showed that ATP induced a biphasic intracellular Ca2+ increase in taste cells by a Ca2+ imaging method. Furthermore, we showed by double-immunolabeling methods that P2Y1-expressing cells coexpressed both IP3R3 and SNAP-25. These results suggest that ATP may activate P2Y receptors resulting in Ca2+ release from internal stores via IP3R3. Since many SNAP-25-immunoreactive taste bud cells coexpressed P2Y1 immunoreactivity, it is suggested that P2Y1-expressing cells may possess synapses with afferent nerve fibers. The results of the present study suggest that P2Y1 receptor may play some roles in ATP-mediated signal transductions between taste bud cells and afferent taste fibers.

Adenosine Triphosphate↗
PubMed2003

Evidence for the presence of nicotinic receptors on rat subfornical organ neurons.

The sensitivity of subfornical organ (SFO) neurons in rat slice preparations to nicotine was studied using whole-cell patch-clamp recordings and immunohistochemistry. In the current-clamp mode, nicotine at 10 microM depolarized the membrane and increased the firing rate. In the voltage-clamp mode, nicotine elicited inward currents in a dose-dependent manner. The net current-voltage relationships of the nicotine-induced currents displayed inward rectification above -40 mV. The relatively alpha4beta2-selective nicotinic acetylcholine (ACh) receptor antagonist, dihydro-beta-erythroidine, reduced the peak amplitudes of the nicotine-induced inward currents. Immunohistochemical experiments with alpha4 antibody showed the existence of immunopositive cells in the subfornical organ. These results suggest that neurons in the subfornical organ are excited via alpha4beta2 nicotinic acetylcholine receptors.

Animals↗
PubMed2003

Expression of metabotropic glutamate receptor group I in rat gustatory papillae.

Taste-metabotropic glutamate receptor 4 (taste-mGluR4) and the heteromers of T1R1 and T1R3 are candidate receptors involved in the sense of umami (monosodium glutamate) taste. Although the expression of group III mGluRs (taste-mGluR4) has been demonstrated in taste tissues, no mention has been made of the expression of group I mGluRs (mGluR1 and mGluR5) in taste tissues. We examined the expression of mGluR1 and mGluR5 in rat gustatory tissues by using reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, immunohistochemistry and immunoelectron microscopy. RT-PCR assay showed that mGluR1 alpha and mGluR1 beta mRNAs were expressed in circumvallate papillae, but mGluR5 mRNA was not expressed. The positive signals of mGluR1 mRNA were detected only in circumvallate taste buds by in situ hybridization analysis. In cryosections of fungiform, foliate and circumvallate papillae, the antibody against mGluR1 alpha gave intense labeling on the taste hairs in all taste pores examined. In the developing taste buds, the positive signals of mGluR1alpha in taste hairs gradually increased with the increase in number of taste bud cells. These results show that, in addition to taste-mGluR4 and the heteromer of T1R1 and T1R3, mGluR1 alpha may function as a receptor for glutamate (umami) taste sensation.

Animals↗
PubMed2002

Epithelial stem cells in teeth.

Many tissues and organs maintain a process known as homeostasis, in which cells are replenished as they die as a result of apoptosis or injury. The continuously growing mouse incisors are an excellent model for studying the molecular mechanisms of cell homeostasis, renewal, and repair. We elucidated these mechanisms in mouse incisors by detecting adult stem cells and analyzing the stem cell lineage by bromodeoxyuridine (BrdU) labeling analysis. The stem cells divide slowly, giving rise to a daughter cell that remains in the cervical loop and a second daughter cell that enters the zone of rapidly dividing inner enamel epithelial cells (transit-amplifying cell population). During subsequent rounds of cell division, the latter cells move toward the incisal end and differentiate into ameloblasts that form the enamel matrix. Recent evidence from gene knockout mice suggests that fibroblast growth factor (Fgf10) plays an important role in the formation and maintenance of stem cells in the development of mouse incisors. The role of dental stem cells in odontogenic tumors is discussed.

Journal Article↗
PubMed2002

Establishment of dental epithelial cell line (HAT-7) and the cell differentiation dependent on Notch signaling pathway.

Rat incisors grow continuously throughout life. Producing a variety of dental epithelial cells is performed by stem cells located in the cervical loop of the incisor apex. To study the mechanisms for cell differentiation, we established a dental epithelial cell line (HAT-7) originating from a cervical loop epithelium of a rat incisor. Immunochemical studies showed that HAT-7 produced the cells expressing amelogenin, ameloblastin, or alkaline phosphatase (ALP). To illustrate a role of Notch signaling in the determinant of the cell fate, we examined expression patterns of Notch1 and Jagged1 in HAT-7 density dependently. At lower cell density, Notch1- or Jagged1-expressing cells were not seen. However, when they were fully confluent, cells began to express Notch1 or Jagged1 strongly. Some ALP-positive cells were almost consistent with Notch1-expressing cells but not Jagged1-expressing cells. These results suggested that the determinant of direction of differentiation was associated with Notch signaling pathway.

Animals↗
PubMed2002

FGF10 maintains stem cell population during mouse incisor development.

Mouse incisors have a cervical loop that gives rise to dental epithelium in the apical region of the tooth germ, in contrast to molars. In a study of formation of the stem-cell compartment, we focused on expression patterns of fibroblast growth factor (Fgf) 10 and Fgf3 in developing mice incisors. At E14, Fgf10 and Fgf3 were coexpressed in the dental papilla. After E16 mesenchymal cells underlying cervical loop expressed Fgf10 but not Fgf3. To illustrate the role of FGF10, we analyzed incisor development of Fgf10-deficient mice. The germs of FGF10-null mice proceeded to cap stage normally. However, at a later stage, the cervical loop was not formed. Functional disorder of FGF10 by a neutralizing anti-FGF10 antibody induced apoptosis in the cervical loop of incisor explants. Recombinant FGF10 rescued the cervical loop from apoptosis. These results show that FGF10 maintains the stem-cell compartment in the developing incisor tooth germ.

Animals↗
PubMed2002

FGF10 maintains stem cell compartment in developing mouse incisors.

Mouse incisors are regenerative tissues that grow continuously throughout life. The renewal of dental epithelium-producing enamel matrix and/or induction of dentin formation by mesenchymal cells is performed by stem cells that reside in cervical loop of the incisor apex. However, little is known about the mechanisms of stem cell compartment formation. Recently, a mouse incisor was used as a model to show that fibroblast growth factor (FGF) 10 regulates mitogenesis and fate decision of adult stem cells. To further illustrate the role of FGF10 in the formation of the stem cell compartment during tooth organogenesis, we have analyzed incisor development in Fgf10-deficient mice and have examined the effects of neutralizing anti-FGF10 antibody on the developing incisors in organ cultures. The incisor germs of FGF10-null mice proceeded to cap stage normally. However, at a later stage, the cervical loop was not formed. We found that the absence of the cervical loop was due to a divergence in Fgf10 and Fgf3 expression patterns at E16. Furthermore, we estimated the growth of dental epithelium from incisor explants of FGF10-null mice by organ culture. The dental epithelium of FGF10-null mice showed limited growth, although the epithelium of wild-type mice appeared to grow normally. In other experiments, a functional disorder of FGF10, caused by a neutralizing anti-FGF10 antibody, induced apoptosis in the cervical loop of developing mouse incisor cultures. However, recombinant human FGF10 protein rescued the cervical loop from apoptosis. Taken together, these results suggest that FGF10 is a survival factor that maintains the stem cell population in developing incisor germs.

Animals↗
PubMed2002

Expression of the metabotropic glutamate receptor, mGluR4a, in the taste hairs of taste buds in rat gustatory papillae.

Taste-mGluR4, cloned from taste tissues, is a truncated variant of brain-expressed mGluR4a (brain-mGluR4), and is known to be a candidate for the receptor involved in the umami taste sense. Although the expression patterns of taste- and brain-mGluR4 mRNAs have been demonstrated, no mention has so far been made of the expression of these two mGluR4 proteins in taste tissues. The present study examined the expression of taste-mGluR4 and brain-mGluR4 proteins in rat taste tissues by using a specific antibody for mGluR4a which shared a C-terminus of both taste- and brain-mGluR4, for immunoblot analysis and immunohistochemistry. Immunoblot analysis showed that both brain-mGluR4 and taste-mGluR4 were expressed in the taste tissues. Taste-mGluR4 was not detected in the cerebellum. The immunoreactive band for brain-mGluR4 protein was much stronger than that for taste-mGluR4 protein. In the cryosections of fungiform, foliate and circumvallate papillae, the antibody against taste-mGluR4 exhibited intense labeling of the taste pores and taste hairs in all the taste buds of gustatory papillae examined; the immunoreaction to the antibody against brain-mGluR4 was more intense at the same sites of the taste buds. The portions of the taste bud cells below the taste pore and surrounding keratinocytes did not show any immunoreactivities. The results of the present study strongly suggest that, in addition to taste-mGluR4, brain-mGluR4 may function even more importantly than the former as a receptor for glutamate, i.e. the umami taste sensation.

Animals↗