哺乳动物舌面味蕾的发育

根据近年来有关大鼠、小鼠味觉发育方面的大量研究,对哺乳动物味蕾(taste buds)发育的情况进行了综述和讨论.哺乳动物舌面上的味蕾分布在菌状乳头(fungiform papillae,FF)、叶状乳头(foliate papillae,FL)、轮廓状乳头(circumvallate papillae,CV)之中,味蕾细胞(taste bud cells)不断地进行着周期性的更新,味蕾的形态、数量和功能随动物随年龄而变化.有关味孔头的研究表明,味乳头(gustatory papillae)在味蕾形成和维持味蕾存在及正常发育方面有着独特的功能.味乳头和味蕾的发育过程与细胞信号分子(signaling molecules)、味觉神经(gustatory nerve fibers)等许多因素有着密切的关系,其中有些作用机理至今尚无定论.     

Oro-Gustatory Perception of Dietary Lipids and Calcium Signaling in Taste Bud Cells Are Altered in Nutritionally Obesity-Prone Psammomys obesus

Since the increasing prevalence of obesity is one of the major health problems of the modern era, understanding the mechanisms of oro-gustatory detection of dietary fat is critical for the prevention and treatment of obesity. We have conducted the present study on Psammomys obesus, the rodent desert gerbil which is a unique polygenic natural animal model of obesity. Our results show that obese animals exhibit a strong preference for lipid solutions in a two-bottle test. Interestingly, the expression of CD36, a lipido-receptor, in taste buds cells (TBC), isolated from circumvallate papillae, was decreased at mRNA level, but remained unaltered at protein level, in obese animals. We further studied the effects of linoleic acid (LA), a long-chain fatty acid, on the increases in free intracellular calcium (Ca²⁺) concentrations, [Ca²⁺]i, in the TBC of P. obesus. LA induced increases in [Ca²⁺]i, largely via CD36, from intracellular pool, followed by the opening of store-operated Ca²⁺ (SOC) channels in the TBC of these animals. The action of this fatty acid on the increases in [Ca²⁺]i was higher in obese animals than that in controls. However, the release of Ca²⁺ from intracellular stores, studied also by employing thapsigargin, was lower in TBC of obese animals than control rodents. In this study, we show, for the first time, that increased lipid intake and altered Ca²⁺ signaling in TBC are associated with obesity in Psammomys obesus.     

The calcium-sensing receptor in taste tissue

Calcium is an essential nutrient that induces a distinctive taste quality, but the sensing mechanism of calcium in the tongue is poorly understood. A recent study linked calcium to T1R3 receptor. Here, we propose another system for calcium taste involving the extracellular calcium-sensing receptor (CaSR). This G protein-coupled receptor that responds to calcium and magnesium cations is involved in calcium homeostasis regulating parathyroid and kidney functions. In this study, CaSR was found in isolated taste buds from rats and mice. It was expressed in a subset of cells in circumvallate and foliate papillae, with fewer cells in the fungiform papillae. This is the first evidence in mammals that locates CaSR in gustatory tissue and provides the basis for better understanding not only calcium taste but also the taste of multiple CaSR agonists.     

The Src family kinase Yes triggers hyperosmotic activation of the epidermal growth factor receptor and CD95

Hyperosmotic exposure of rat hepatocytes triggers epidermal growth factor receptor (EGFR) activation, which results in an activation of the CD95 system and sensitizes the cells toward apoptosis (Reinehr, R., Schliess, F., and Haüssinger, D. (2003) FASEB J. 17, 731-733). The mechanisms underlying the hyperosmotic EGFR activation were studied. Hyperosmotic exposure (405 mosm) resulted in a rapid activation of the Src kinase family members Yes, Fyn, and Lck. Hyperosmotic Yes, but not Fyn activation, was antioxidant-sensitive and was followed by a rapid Yes/EGFR association. PP-2 abolished the hyperosmotic activation of Fyn and Lck but not activation of Yes and EGFR and their association. However, these latter processes were prevented in the presence of SU6656. SU6656 and antioxidants, but not PP-2 and AG1478, also inhibited the hyperosmotic JNK activation. Cyclic AMP had no effect on hyperosmotic Yes and JNK activation but prevented EGFR/Yes association and EGFR activation in an H89-sensitive way. When the hyperosmolarity-induced Yes-EGFR protein complex started to disappear after 30 min, an association between EGFR and CD95 became apparent, which was followed by CD95 tyrosine phosphorylation and activation. SU6656 but not PP-2 also inhibited EGFR/CD95 association, CD95 tyrosine phosphorylation, CD95 membrane trafficking, and death-inducing signaling complex (DISC) formation. EGFR knockdown had no effect on hyperosmotic Yes activation but prevented CD95 tyrosine phosphorylation, membrane targeting, and DISC formation. Hyperosmotic EGFR and CD95 activation was also largely blunted following Yes knockdown. The data suggest that hyperosmotic signaling triggers an oxidative stress-dependent Yes activation, which is followed by JNK and EGFR activation and subsequent activation of the CD95 system. However, the functional relevance of hyperosmolarity-induced Fyn and Lck activation remains to be elucidated.     

Expression of tryptophan hydroxylase in developing mouse taste papillae

Gustatory papillae and associated taste buds receive and process chemical information from the environment. In mammals, their development takes place during the late phase of embryogenesis. However, the cellular factors that regulate the differentiation of taste papillae remain largely unknown. Here, we show by quantitative real time RT-PCR that both isoforms of tryptophan hydroxylase (TPH1 and TPH2), the first and rate limiting enzyme of serotonin (5-HT) synthesis, are expressed in developing circumvallate papillae. Immuno-staining experiments further indicated that TPH is localized both in gustatory fibers and in differentiated taste receptor cells. These results point to the synthesis of 5-HT in gustatory papillae, and allow one to hypothesize that the development of taste buds might be modulated by serotonin.     

Fyn binds to and phosphorylates the kidney slit diaphragm component Nephrin

Recent investigations have focused on characterizing the molecular components of the podocyte intercellular junction, because several of these components, including Nephrin, are functionally necessary for development of normal podocyte structure and filter integrity. Accumulating evidence suggests that the Nephrin-associated protein complex is a signaling nexus. As such, Nephrin-dependent signaling might be mediated in part through Nephrin phosphorylation. Described are biochemical and mouse genetics experiments demonstrating that membrane-associated Nephrin is tyrosine-phosphorylated by the Src family kinase Fyn. Nephrin fractionated in detergent-resistant glomerular membrane fractions with Fyn and Yes. Fyn directly bound Nephrin via its SH3 domain, and Fyn directly phosphorylated Nephrin. Glomeruli in which Fyn, Yes, or Fyn and Yes were genetically deleted in mice were characterized to explore the relationship between these kinases and Nephrin. Fyn deletion resulted in coarsening of podocyte foot processes and marked attenuation of Nephrin phosphorylation in isolated glomerular detergent-resistant membrane fractions. Yes deletion had no identifiable effect on podocyte morphology but dramatically increased Nephrin phosphorylating activity. Similar to Fyn deletion, simultaneous deletion of Fyn and Yes reduced Nephrin phosphorylating activity. These results demonstrate that endogenous Fyn catalyzes Nephrin phosphorylation in podocyte detergent-resistant membrane fractions. Although Yes appears to effect the regulation of Nephrin phosphorylation, the mechanism by which this occurs requires investigation.     

Chronic hypoxia alters calbindin D-28k immunoreactivity in lingual and laryngeal taste buds in the rat

The distribution and abundance of the calcium binding protein, calbindin D-28k (CB) immunoreactivity in the taste buds of the circumvallate papillae and larynx were compared between normoxic and chronically hypoxic rats (10% O2 for 8 weeks). In the normoxic rats, CB immunoreactivity was observed in some cells and fibers of the intragemmal region of the taste buds in the circumvallate papillae. In contrast, in the subgemmal region of the laryngeal taste buds, fibers but not cells were immunoreactive for CB. In chronically hypoxic rats, CB immunoreactive cells and fibers in the taste buds were decreased in the circumvallate papillae. In the laryngeal taste buds, the density of the subgemmal CB immunoreactive fibers in chronically hypoxic rats was greater than in normoxic rats. It is considered that function of the laryngeal taste buds is different from that of the lingual taste buds, so that laryngeal taste buds may be involved in chemosensation other than taste. The altered density of CB immunoreactive cells and fibers in the lingual and laryngeal taste buds is a predominant feature of hypoxic adaptation, and chronic hypoxic exposure might change the chemical sensitivity of the circumvallate papillae and larynx through the regulation of intracellular Ca2+.     

Spacing patterns on tongue surface-gustatory papilla

The dorsal surface of the mammalian tongue is covered with four kinds of papillae, fungiform, circumvallate, foliate and filiform papillae. With the exception of the filiform papillae, these types of papillae contain taste buds and are known as the gustatory papillae. The gustatory papillae are distributed over the tongue surface in a distinct spatial pattern. The circumvallate and foliate papillae are positioned in the central and lateral regions respectively and the fungiform papillae are distributed on the anterior part of the tongue in a stereotyped array. The patterned distribution and developmental processes of the fungiform papillae indicate some similarity between the fungiform papillae and the other epithelial appendages, including the teeth, feathers and hair. This is because 1) prior to the morphological changes, the signaling molecules are expressed in the fungiform papillae forming area with a stereotyped pattern; 2) the morphogenesis of the fungiform papillae showed specific structures in early development, such as epithelial thickening and mesenchymal condensation and 3) the fungiform papillae develop through reciprocal interactions between the epithelium and mesenchymal tissue. These results led us to examine whether or not the early organogenesis of the fungiform papillae is a good model system for understanding both the spacing pattern and the epithelial-mesenchymal interaction during embryogenesis.     

Induction of apoptosis by colchicine in taste bud and epithelial cells of the mouse circumvallate papillae

Apoptotic cells in the taste buds and epithelia of mouse circumvallate papillae after colchicine treatment were examined by the methods of in situ DNA nick-end labeling, immunocytochemistry, and electron microscopy. After colchicine treatment, numerous positive cells appeared in the taste buds by DNA nick-end labeling, and some epithelial cells in the basal and suprabasal layers in and around the circumvallate papillae also revealed positive staining. Condensed and fragmented nuclei with a high density were occasionally found in the taste bud cells and in the basal and suprabasal layer epithelial cells by electron-microscopic observation. An immunocytochemical reaction for tubulin revealed weak staining in taste bud cells, because of the depolymerization of microtubules, and a decrease of the microtubules in the taste bud cells was observed by electron microscopy. These results indicate that colchicine treatment of mice induces the apoptosis of taste bud and epithelial cells in the circumvallate papillae and dorsal epithelial cells around the circumvallate papillae.     

Immunohistochemical localization of neuron-specific enolase and calcitonin gene-related peptide in rat taste papillae.

Calcitonin gene-related peptide-like and neuron-specific enolase-like immunoreactivity (CGRP-IR and NSE-IR) were surveyed immunohistochemically in the fungi-form, foliate and circumvallate papillae in rats. A dense CGRP-IR network (subgemmal and extragemmal) in the taste papillae is linked to the presence of taste buds, even though CGRP-IR fibers are rarely present in the taste buds. Three typical fiber populations were detected with these two markers. (a) A population of coarse NSE-IR intragemmal fibers characterized by thick neural swellings, never expressing CGRP-immunoreactivity. (b) A population of thin varicose intragemmal NSE/CGRP-IR fibers. (c) A population of subgemmal and extragemmal NSE-/CGRP-IR fibers that partly penetrated the epithelium. The common distribution of CGRP-IR and NSE-IR fibers at the base of taste buds, their differential distribution and morphology within taste buds, added to their restricted nature (gustatory or somatosensory) suggest that a population of CGRP-IR fibers undergoes a target-induced inhibition of its CGRP phenotype while entering the taste buds. The combined use of NSE and CGRP allowed a better characterization of nerve fibers within and between all three types of taste papillae. NSE was also a very good marker for a subtype of taste bud cells in the foliate and in the circumvallate papillae, but no such cells could be observed in the fungiform papillae.     

Immunohistochemical localization of carbonic anhydrase isozyme II in the gustatory epithelium of the adult rat.

The distribution of carbonic anhydrase isozyme II (CA II)-like immunoreactivity (-LI) in the gustatory epithelium was examined in the adult rat. In the circumvallate and foliate papillae, CA II-LI was observed in the cytoplasm of the spindle-shaped taste bud cells, with weak immunoreaction in the surface of the gustatory epithelium. No neuronal elements displayed CA II-LI in these papillae. There was no apparent difference in the distribution pattern between the anterior and posterior portions of the foliate papillae. In immunoelectron microscopy, immunoreaction products for CA II were diffusely distributed in the entire cytoplasm of the taste bud cells having dense round granules at the periphery of the cells. No taste bud cells displaying CA II-LI were detected in the fungiform papillae, but a few thick nerve fibers displayed CA II-LI. In the taste buds of the palatal epithelium, neither taste bud cells nor neuronal elements exhibited CA II-LI. The present results indicate that CA II was localized in the type I cells designated as supporting cells in the taste buds located in the posterior lingual papillae of the adult animal.     

Expression of cyclin-dependent kinase inhibitors in taste buds of mouse and hamster

Taste buds are specialized epithelial cell clusters in the oral squamous cell epithelium. Although taste buds have been reported to renew rapidly, the mechanism of cell cycle control in these specialized structures remains unresolved. To clarify the cell cycle status and role of cyclin-dependent kinase inhibitors (CDKI) for cell cycle control in the taste buds, we analyzed cell proliferation activity using bromodeoxyuridine (BrdU) and Ki-67 immunostainings and the expression of the Cip/Kip family of CDKI (p21Cip1, p27Kip1, and p57Kip2) in the circumvallate papillae of mouse and hamster. BrdU-positive cells were detected in the basal layer of the oral epithelium. In the taste buds, Ki-67-positive cells were seen in the basal area, with only a very few positive cells in the taste buds. Both p21Cip1 and p27Kip1 positive cells were seen in the suprabasal layer of the non-gustatory oral epithelium. In the taste buds, stronger p27Kip1 staining was detected than in the non-gustatory epithelium. Western blotting analysis revealed that p27Kip1 was abundant in the mucosal tissues from circumvallate papillae. Thus, our study suggests that the taste bud cells except for basal cells are post-mitotic cells and that the cell cycle arrest associated with taste bud cell differentiation could be regulated predominantly by p27Kip1.     

Blood vascular architecture of the rat lingual papillae with special reference to their relations to the connective tissue papillae and surface structures: a light and scanning electron microscope study

Subepithelial blood vessels of the rat lingual papillae and their spatial relations to the connective tissue papillae and surface structures were demonstrated by light and scanning electron microscopy. In the rat, four types of papillae were distinguished on the dorsal surface of the tongue, i.e. the filiform, fungiform, foliate and circumvallate papillae. Vascular beds of various appearance were found in all four types of lingual papillae: a simple or twisted capillary loop in the filiform papilla; a basket- or petal-like network in the fungiform papilla; a ring-like network in the foliate papilla, and a conglomerated network surrounded by double heart-shaped capillary networks in the circumvallate papilla. These characteristic vascular beds corresponded to the shape of the connective tissue papillae and surface structures. The vascular bed beneath the gustatory epithelium in the fungiform, foliate and circumvallate papilla consisted of fine capillary networks next to the taste buds.     

Comparative phosphoproteomics of zebrafish Fyn/Yes morpholino knockdown embryos

The coordinated movement of cells is indispensable for normal vertebrate gastrulation. Several important players and signaling pathways have been identified in convergence and extension (CE) cell movements during gastrulation, including non-canonical Wnt signaling. Fyn and Yes, members of the Src family of kinases, are key regulators of CE movements as well. Here we investigated signaling pathways in early development by comparison of the phosphoproteome of wild type zebrafish embryos with Fyn/Yes knockdown embryos that display specific CE cell movement defects. For quantitation we used differential stable isotope labeling by reductive amination of peptides. Equal amounts of labeled peptides from wild type and Fyn/Yes knockdown embryos were mixed and analyzed by on-line reversed phase TiO(2)-reversed phase LC-MS/MS. Phosphorylated and non-phosphorylated peptides were quantified, and significant changes in protein expression and/or phosphorylation were detected. We identified 348 phosphoproteins of which 69 showed a decrease in phosphorylation in Fyn/Yes knockdown embryos and 72 showed an increase in phosphorylation. Among these phosphoproteins were known regulators of cell movements, including Adducin and PDLIM5. Our results indicate that quantitative phosphoproteomics combined with morpholino-mediated knockdowns can be used to identify novel signaling pathways that act in zebrafish development in vivo.     

Apoptosis in mouse taste buds after denervation

Apoptotic cells in the taste buds of mouse circumvallate papillae after the sectioning of bilateral glossopharyngeal nerves were examined by the method of DNA nick-end labeling (TUNEL), together with standard electron microscopy. The taste buds decreased in number and size 3–11 days after denervation and disappeared at 11 days. The TUNEL method revealed only a few positively stained nuclei in normal taste buds but, in those of mice 1–5 days after denervation, the number of positive nuclei had increased to 3–5 times that of taste buds from normal mice. Electron-microscopic observation after denervation demonstrated taste bud cells containing condensed and fragmentary nuclei in a cytoplasm with increased density. The results show that taste bud cells under normal conditions die by apoptosis at the end of their life span, and that gustatory nerve sectioning causes apoptosis of taste bud cells with taste buds decreasing in number and ultimately disappearing. Received: 20 November 1995 / Accepted: 15 May 1996     

Histochemical observations of alkaline phosphatase activity of the lingual epithelium after the suppression of salivation

Summary The influence of salivation on the location of gustatory alkaline phosphatase has been examined. In untreated rats, taste buds at the ends of fungiform papillae showed almost no activity. However, if salivation was suppressed for 12 hours in fasted animals, alkaline phosphatase activity could be clearly demonstrated in association with these taste buds. The results indicated that alkaline phosphatase may be removed from its site of secretion by saliva and that the enzyme is secreted from fungiform as well as circumvallate and foliate papillae.     

Src family kinases are required for integrin but not PDGFR signal transduction

Src family kinases (SFKs) have been implicated as important regulators of ligand-induced cellular responses including proliferation, survival, adhesion and migration. Analysis of SFK function has been impeded by extensive redundancy between family members. We have generated mouse embryos harboring functional null mutations of the ubiquitously expressed SFKs Src, Yes and Fyn. This triple mutation leads to severe developmental defects and lethality by E9.5. To elucidate the molecular mechanisms underlying this phenotype, SYF cells (deficient for Src, Yes and Fyn) were derived and tested for their ability to respond to growth factors or plating on extracellular matrix. Our studies reveal that while Src, Yes and Fyn are largely dispensable for platelet-derived growth factor (PDGF)-induced signaling, they are absolutely required to mediate specific functions regulated by extracellular matrix proteins. Fibronectin-induced tyrosine phosphorylation of focal adhesion proteins, including the focal adhesion kinase FAK, was nearly eliminated in the absence of Src, Yes and Fyn. Furthermore, consistent with previous reports demonstrating the importance of FAK for cell migration, SYF cells displayed reduced motility in vitro. These results demonstrate that SFK activity is essential during embryogenesis and suggest that defects observed in SYF triple mutant embryos may be linked to deficiencies in signaling by extracellular matrix-coupled receptors.