The reversible effect of colchicine on the taste bud cells of the central circumvallate papilla in the rat

Summary It is believed that differentiation and maintenance of taste buds in vertebrates is dependent on the trophic function of their sensory nerve supply. In the present work colchicine was injected into the circumvallate papilla of the rat. This produced a reversible blockade of neuroplasmic transport and disappearance of taste buds. Colchicine inhibited the further differentiation of bud cells, but apparently did not change the life cycle of the cells present already at the time of injection. It is speculated that the neurotrophic factors in this particular cell system are effective to induce cell differentiation only.This work was supported by CAIT Grant No 1776     

Freeze-fracture study of taste bud pores in the foliate papillae of the rabbit

Summary In freeze-fractured specimens of taste buds from the foliate papillae of rabbits, the intercellular spaces are separated from the pore of the taste bud by zonulae occludentes of the tight-type. Below these tight junctions numerous desmosomes are found at irregular intervals. The epithelial cells adjacent to the pore are also joined by single strands of fusion. The microvilli arising from the neck of the type I cells have a high particle density. The microvilli of type II cells and especially the short microvilli of peripherally situated cells have a lower intramembranous particle density. The single microvillus of type III cells has a very large diameter and is longer than the other microvilli. It contains a few larger intramembranous particles and vesicle-like protrusions of the membrane facing the cytoplasm. Transverse fracturing reveals a filamentous fine structure in all microvilli. The physiological implications of these observations are discussed.Supported by grants from the Deutsche Forschungsgemeinschaft (Ja 205/5+6)     

Role of calcium signaling in epithelial bicarbonate secretion

Transepithelial bicarbonate secretion plays a key role in the maintenance of fluid and protein secretion from epithelial cells and the protection of the epithelial cell surface from various pathogens. Epithelial bicarbonate secretion is mainly under the control of cAMP and calcium signaling. While the physiological roles and molecular mechanisms of cAMP-induced bicarbonate secretion are relatively well defined, those induced by calcium signaling remain poorly understood in most epithelia. The present review summarizes the current status of knowledge on the role of calcium signaling in epithelial bicarbonate secretion. Specifically, this review introduces how cytosolic calcium signaling can increase bicarbonate secretion by regulating membrane transport proteins and how it synergizes with cAMP-induced mechanisms in epithelial cells. In addition, tissue-specific variations in the pancreas, salivary glands, intestines, bile ducts, and airways are discussed. We hope that the present report will stimulate further research into this important topic. These studies will provide the basis for future medicines for a wide spectrum of epithelial disorders including cystic fibrosis, Sjögren's syndrome, and chronic pancreatitis.     

Collection of gametes from live axolotl, Ambystoma mexicanum, and standardization of in vitro fertilization

This study established the first protocol for collection of gametes from live axolotl, Ambystoma mexicanum, by gentle abdominal massage and in vitro fertilization. To stimulate spermiation and ovulation, human chorionic gonadotrophin (hCG) and Ovopel pellets, which are commercially used to stimulate spawning in fish, were tested. The hCG was more effective than Ovopel pellets and yielded a higher semen volume in the injected males and a shorter response time in the females. Collected semen by this method was already motile and fertile. Fertile eggs could be collected in 3-4 successive collection times after the female has started the typical spawning behaviour. The fertilization condition that yielded the highest hatching rate was mixing semen with eggs before the addition of a fertilization saline solution (20 mmol/l NaCl, 1 mmol/l KCl, 1 mmol/l Mg₂SO₄, 1 mmol Ca₂Cl, 3 mmol NaHCO₃, 10 mmol/l Tris, pH 8.5 - Osmolality = 65 mosmol/kg). When the pH of the fertilization solution was increased to ≥ 10, the hatching rate was significantly increased. The use of fertilization solutions with osmolalities of ≥ 150 and ≥ 182 were accompanied with a significant decrease in hatching rates and the appearance of deformed larvae, respectively. In conclusion, a reliable protocol for gamete collection from live axolotl is established as a laboratory model of in vitro fertilization for urodele amphibians. This protocol may be transferable to endangered urodeles.     

Synaptophysin as a probable component of neurotransmission occurring in taste receptor cells

Taste signal is received in taste buds and transmitted via sensory afferent nerves to the brainstem. Although a signaling pathway involving phospholipase C-β2 has been shown to transduce taste signals of bitterness, sweetness and umami in taste receptor cells (Type II cells), these taste receptor cells appear to be different from the presynaptic cells (Type III cells) containing afferent synapses associated with nerve processes. To elucidate the neurotransmission system in the taste receptor cells expressing phospholipase C-β2, we searched for candidate molecules involved in the neurotransmission, and identified synaptophysin. Synaptophysin was expressed in the taste receptor cells expressing phospholipase C-β2, as well as in the presynaptic cells harboring synaptic structures with taste nerves and containing serotonin. Synaptophysin-immunoreactive signals were not limited to gustducin-positive bitter taste receptor cells, and sweet/umami taste receptor cells were indicated to also express synaptophysin. Expression of synaptophysin was already initiated 6 days after cell division, almost in synchrony with the initiation of phospholipase C-β2 expression. Synaptophysin-containing cells co-expressed vesicular-associated membrane protein 2, a v-SNARE molecule which is important for exocytosis. In addition, majority of the synaptophysin-expressing cells also expressed cholecystokinin, a neuropeptide expressed in taste buds. These results suggest that the taste receptor cells have a neurotransmission system involving synaptophysin, which occurs alternatively or additionally to a recently shown hemichannel system.     

Ultrastructure and distribution of the taste buds in the buccal cavity in relation to the food and feeding habit of a herbivorous fish: Oreochromis niloticus

The buccal cavity of an herbivorous fish Oreochromis niloticus was investigated by means of scanning electron microscopy. The buccal cavity distinguished into the roof and the floor. Three different types of taste buds (type I, II and III) are distributed in the buccal cavity. The proximal part of the buccal cavity bears relatively high epidermal papillae in which type I TBs was found. The middle region of the buccal cavity is mostly occupied by low epidermal papillae containing type II TBs. Type III TBs which are present within the metabranchial buccal cavity; never rise above the normal level of the epithelium.The different types of TBs are useful for ensuring full utilization of the gustatory ability of the fish. It is postulated that the TBs serve different functions: TBs type I and II may act as chemoreceptors and mechanoreceptors. TBs type III acts predominantly as a chemoreceptors. TBs of each type show two kinds of receptor villi within their receptor areas: tall villi and small villi. The surface of the lining epithelial cells shows a delicate microridge system. These structures protect against physical abrasion potentially caused during food maneuvering and swallowing. Furthermore, protection of the epithelium from abrasion is enhanced with goblet cells secretion.     

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.     

肺内调节肽对兔支气管上皮细胞分泌白介素的影响

为探讨肺内调节肽对气管上皮细胞（brochial epithelial cells,BECs）分泌功能的影响,实验观察了兔BECs在未受应激与臭氧应激两种条件下白细胞介素（interleukin,ILs）的分泌。结果发现：血管活性肠肽（vasoac-tive intestinal peptide,VIP）以未受应激BECs存在抑制作用,并使用臭氧应激BECs分泌ILs下降;表皮生长因子（epi-dermal growth factor,EGF）使未受应激BECs IL-1、IL-8分泌增加,使臭氧应激的BECs ILs分泌降低;内皮素-1（endothelin-1,ET-1）、降钙素基因相关肽(calctionin gene-related peptide,CGRP）可使未受应激的BECs分泌ILs增加,CGRP还可使臭氧应激的BECs ILs分泌增加。结果提示：肺内调节肽可调控BECs ILs的分泌,在调控气道炎症损伤信号传递方面具有一定的作用。     

Demonstration and maintenance of mucus secretion in cultured human gallbladder epithelial cells

Summary The method of human gallbladder epithelial cell culture has been developed successfully with active mucus secretory function. Human gallbladder epithelial cells were dissociated by Dispase digestion from the specimens obtained by cholecystectomy for uncomplicated gallbladder stone cases. The dissociated cells formed a monolayer in Eagle’fs minimum essential medium supplemented with 10% fetal bovine serum within 24 h after the inoculation. These cells were maintained for at least 2 wk without fibroblastic overgrowth. Cultured cells contained periodic acid Schiff-positive material in cellular cytoplasm for 3 d. On transmission electron microscopy these materials were identified as mucous secretory granules. Mucous secretory function was determined by [³H]glucosamine incorporation. Sixty percent of the secreted glycoproteins labeled with [³H]glucosamine was eluted in excluded fractions of Sepharose 4B gel filtration, which were considered to be mucous glycoprotein, because they were found to be resistant to proteoglycan-specific enzymes such as hyaluronidase, chondroitinase ABC, heparitinase, and heparinase. The mucous glycoprotein secretion was maintained for 3 d and found to be inhibited in a dose-dependent manner by monensin (10⁻⁷ to 10⁻⁵ M) which is a known blocker of secretory function.     

Responses of MSH and prolactin cells to 5-hydroxytryptophan (5-HTP) in amphibians and teleosts

Summary Injection of 5-hydroxytryptophan (5-HTP), a precursor of serotonin, induces dispersion of melanin in the amphibians, Pleurodeles waltlii (Urodela) and Xenopus laevis (Anura), in the goldfish, Carassius auratus, and in the carp, Cyprinus carpio. It is accompanied by a dispersion of erythrophore pigments. In the pituitaries of Pleurodeles and goldfish, a stimulation of MSH cells, characterized by a significant nuclear hypertrophy, is also observed; in Carassius, MSH cells may become degranulated. Serotonin appears to exert a stimulating effect on MSH release in lower vertebrates. Swimming behavior is disturbed in the goldfish and the carp; gaseous metabolism in the swim-bladder may be affected by injection of 5-HTP, as previously reported in the eel. Prolactin (PRL) cells appear activated, but remain granulated in the treated goldfish. No clear response of PRL cells to injection of 5-HTP can be observed in Pleurodeles. A possible role of serotonin in Pleurodeles submitted to an experimental aeroionization is briefly discussed.     

Multiple Shh signaling centers participate in fungiform papilla and taste bud formation and maintenance

The adult fungiform taste papilla is a complex of specialized cell types residing in the stratified squamous tongue epithelium. This unique sensory organ includes taste buds, papilla epithelium and lateral walls that extend into underlying connective tissue to surround a core of lamina propria cells. Fungiform papillae must contain long-lived, sustaining or stem cells and short-lived, maintaining or transit amplifying cells that support the papilla and specialized taste buds. Shh signaling has established roles in supporting fungiform induction, development and patterning. However, for a full understanding of how Shh transduced signals act in tongue, papilla and taste bud formation and maintenance, it is necessary to know where and when the Shh ligand and pathway components are positioned. We used immunostaining, in situ hybridization and mouse reporter strains for Shh, Ptch1, Gli1 and Gli2-expression and proliferation markers to identify cells that participate in hedgehog signaling. Whereas there is a progressive restriction in location of Shh ligand-expressing cells, from placode and apical papilla cells to taste bud cells only, a surrounding population of Ptch1 and Gli1 responding cells is maintained in signaling centers throughout papilla and taste bud development and differentiation. The Shh signaling targets are in regions of active cell proliferation. Using genetic-inducible lineage tracing for Gli1-expression, we found that Shh-responding cells contribute not only to maintenance of filiform and fungiform papillae, but also to taste buds. A requirement for normal Shh signaling in fungiform papilla, taste bud and filiform papilla maintenance was shown by Gli2 constitutive activation. We identified proliferation niches where Shh signaling is active and suggest that epithelial and mesenchymal compartments harbor potential stem and/or progenitor cell zones. In all, we report a set of hedgehog signaling centers that regulate development and maintenance of taste organs, the fungiform papilla and taste bud, and surrounding lingual cells. Shh signaling has roles in forming and maintaining fungiform papillae and taste buds, most likely via stage-specific autocrine and/or paracrine mechanisms, and by engaging epithelial/mesenchymal interactions.     

Changes in the cell population of taste buds during degeneration and regeneration of their sensory innervation

Summary Taste buds of rabbit foliate papillae were observed in control, after denervation and during reinnervation by the glossopharyngeal nerve. In control, taste bud cells could be divided into three groups according to their shapes and staining characteristics. Most of the cells were identified as either dark (corresponding to gustatory) or light (corresponding to supporting) cells. However, some cells were encountered which could not readily be placed in either group; they have been termed intermediate cells. Nine to twelve hours after axotomy, wandering cells were observed in many of the taste buds. Thereafter taste buds gradually decreased in size and disappeared, for the most part, by the 14th postoperative day. It was found that dark cells disappeared first, then at a later stage the light cells also disappeared. During reinnervation, dark cells were first to appear about 40 days after the operation and light cells were not seen till about 9 days later.From the observations, it is concluded that the dark cells of the taste bud differentiate from epithelial cells under the influence of nerves and mature into light cells through intermediate cells.     

IL13 activates autophagy to regulate secretion in airway epithelial cells

Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent.     

Gustatory papillae and taste bud development and maintenance in the absence of TrkB ligands BDNF and NT-4

Taste buds and the peripheral nerves innervating them are two important components of the peripheral gustatory system. They require appropriate connections for the taste system to function. Neurotrophic factors play crucial roles in the innervation of peripheral sensory organs and tissues. Both brain-derived neurotrophic factor (BDNF) null-mutated and neurotrophin-4 (NT-4) null-mutated mice exhibit peripheral gustatory deficits. BDNF and NT-4 bind to a common high affinity tyrosine kinase receptor, TrkB (NTRK-2), and a common p75 neurotrophin receptor (NGFR). We are currently using a transgenic mouse model to study peripheral taste system development and innervation in the absence of both TrkB ligands. We show that taste cell progenitors express taste cell markers during early stages of taste bud development in both BDNF^−/−xNT-4^−/− and wild-type mice. At early embryonic stages, taste bud progenitors express Troma-1, Shh, and Sox2 in all mice. At later stages, lack of innervation becomes a prominent feature in BDNF^−/−xNT-4^−/− mice leading to a decreasing number of fungiform papillae and morphologically degenerating taste cells. A total loss of vallate taste cells also occurs in postnatal transgenic mice. Our data indicate an initial independence but a later permissive and essential role for innervation in taste bud development and maintenance. This work was supported by NIH-NIDCD R01-RDC007628.     

Carbenoxolone-sensitive and cesium-permeable potassium channel in the rod cells of frog taste discs

The rod cells in frog taste discs display the outward current and maintain the negative resting potential in the condition where internal K⁺ is replaced with Cs⁺. We analyzed the properties of the Cs⁺-permeable conductance in the rod cells. The current–voltage (I/V) relationships obtained by a voltage ramp were bell-shaped under Cs⁺ internal solution. The steady state I/V relationships elicited by voltage steps also displayed the bell-shaped outward current. The activation of the current accelerated with the depolarization and the inactivation appeared at positive voltage. The gating for the current was maintained even at symmetric condition (Cs⁺ external and internal solutions). The wing cells did not show the properties. The permeability for K⁺ was a little larger than that for Cs⁺. Internal Na⁺ and NMDG⁺ could not induce the bell-shaped outward current. Carbenoxolone inhibited the bell-shaped outward Cs⁺ current dose dependently (IC₅₀: 27 μM). Internal arachidonic acid (20 μM) did not induce the linear current–voltage (I–V) relationship which is observed in two-pore domain K⁺ channel (K_2P). The results suggest that the resting membrane potentials in the rod cells are maintained by the voltage-gated K⁺ channels.     

Cholesterol-dependent regulation of adenosine A2A receptor-mediated anion secretion in colon epithelial cells

Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. In this study we have investigated the role of cholesterol in the adenosine-dependent regulation of ion transport in colonic epithelial cells. We observed that methyl-β-cyclodextrin (MβCD), a cholesterol-sequestering molecule, enhanced adenosine A_2A receptor-activated transepithelial short circuit current (I_sc), but only from the basolateral side. Cholesterol is a major constituent of membrane microdomains, called lipid rafts that also contain sphingolipids. However, studies with the sphingomyelin-degrading enzyme, sphingomyelinase, and the cholesterol-binding agent, filipin, indicated that the change in the level of cholesterol alone was sufficient to control the adenosine-modulated I_sc. Cholesterol depletion had a major effect on the functional selectivity of A_2A receptors. Under control conditions, adenosine activated I_sc more potently than the specific A_2A agonist, CGS-21680, and the current was inhibited by XE991, an inhibitor of cAMP-dependent K⁺ channels. Following cholesterol depletion, CGS-21680 activated I_sc more potently than adenosine, and the current was inhibited by clotrimazole, an inhibitor of Ca²⁺-activated K⁺ (IK1) channels. Co-immunoprecipitation experiments revealed that A_2A receptors associate with IK1 channels following cholesterol depletion. These results suggest that cholesterol content in colonic epithelia affects adenosine-mediated anion secretion by controlling agonist-selective signaling.