Taste Disks are Induced in the Lingual Epithelium of Salamanders during Metamorphosis

Morphological changes of oral cavity during metamorphosis withspecial reference to the taste organ were examined in Ezo salamanders(Hynobius retardatus) and axolotls (Ambystoma mexicanum), andcompared with those in bullfrogs (Rana catesbeiana). The non-distensibletongue of salamanders changed the structure progressively duringmetamorphosis: a small area of the rostrum protruded and developedcaudally with recession of the flat area of the tongue. Theprotrusion that developed on the tongue had numerous papillae,as seen in the frog tongue. The apical region of the papillaeoccasionally had a cell mass similar to the taste disk of frogs(termed a taste disk-like cell mass). On the flat area of thetongue, the barrel-shaped taste buds of larval salamanders weretransformed into taste buds with a wider receptor area. Thebarrel-shaped taste buds decreased progressively during metamorphosis,while taste disk-like cell masses increased. Neuronal labelingwith an antibody to neuron-specific enolase and fluorescentcarbocyanine dye showed that the taste disk-like cell massesin metamorphosed salamanders were innervated by the glossopharyngealnerve (nerve IX). Nerve IX responded to taste stimulation aswell as mechanical stimulation applied to the rostral tongue.During metamorphosis the salamanders undergo transformationand rearrangement of taste organs on the tongue possibly asan adaptation to the terrestrial environment. Chem. Senses 22:535–545, 1997.     

Pharmaco-histochemical studies on a specific monoamine in the gustatory epithelia of the rabbit

Summary The foliate, vallate and fungiform papillae of the rabbit's tongue were studied fluorescence-histochemically under normal and experimental conditions. In normal animals a yellow fluorescence suggesting the presence of a serotonin-like monoamine was demonstrated only in taste bud cells of the foliate papilla, though its intensity was very weak. The fluorescence disappeared completely following reserpine treatment, while it was significantly enhanced by the treatment with nialamide. The fluorescence of taste bud cells could be clearly distinguished from that of catecholamines by the treatment with -MMT followed by nialamide. When 5-HTP, 5-HT and 5,6-DHT were administered separately, each of these drugs was selectively taken up in taste bud cells of the foliate and vallate papillae, but no fluorescent cells were observed in the fungiform papilla.From the present results, it seems reasonable to conclude that the fluorigenic amine of taste bud cells may be 5-HT (serotonin), or at least an indoleamine derivative. Also, it is suggested that the taste bud of the vallate papilla contains a cell type which can potentially synthesize a biogenic amine in situ, or is actually synthesizing it in a very small amount just like in the case of the taste bud of the foliate one.     

Pharmaco-histochemical studies on a specific monoamine in the gustatory epithelia of the rabbit.

The foliate, vallate and fungiform papillae of the rabbit's tongue were studied fluorescence-histochemically under normal and experimental conditions. In normal animals a yellow fluorescence suggesting the presence of a serotonin-like monoamine was demonstrated only in taste bud cells of the foliate papilla, though its intensity was very weak. The fluorescence disappeared completely following reserpine treatment, while it was significantly enhanced by the treatment with nialamide. The fluorescence of taste bud cells could be clearly distinguished from that of catecholamines by the treatment with alpha-MMT followed by nialamide. When 5-HTP, 5-HT and 5,6-DHT were administered separately, each of these drugs was selectively taken up in taste bud cells of the foliate and vallate papillae, but no fluorescent cells were observed in the fungiform papilla. From the present results, it seems reasonable to conclude that the fluorigenic amine of taste bud cells may be 5-HT (serotonin), or at least an indoleamine derivative. Also, it is suggested that the taste bud of the vallate papilla contains a cell type which can potentially synthesize a biogenic amine in situ, or is actually synthesizing it in a very small amount just like in the case of the taste bud of the foliate one.     

Location of taste buds in intact taste papillae by a selective staining method.

Taste buds were found to stain strongly and selectively in intact papillae with highly acidic dyes such as ponceau S. In intact tongues the taste buds in the fungiform, circumvallate and foliate papillae of the cynomolgus monkey and in the fungiform papillae of the rat as well as the taste discs in the fungiform papillae of the frog could be visualized. This method enables a rapid location and counting of taste buds in taste papillae without preparing histological sections. In cynomolgus tongue material fixed in formalin, the dyes penetrate into the buds. In fresh tongues only the taste pore region of the buds stains, which suggests that in vivo taste buds are impenetrable underneath the pore.     

Location of taste buds in intact taste papillae by a selective staining method

Summary Taste buds were found to stain strongly and selectively in intact papillae with highly acidic dyes such as ponceau S. In intact tongues the taste buds in the fungiform, circumvallate and foliate papillae of the cynomolgus monkey and in the fungiform papillae of the rat as well as the taste discs in the fungiform papillae of the frog could be visualized. This method enables a rapid location and counting of taste buds in taste papillae without preparing histological sections. In cynomolgus tongue material fixed in formalin, the dyes penetrate into the buds. In fresh tongues only the taste pore region of the buds stains, which suggests that in vivo taste buds are impenetrable underneath the pore.     

Non-synaptic transformation of gustatory receptor potential by stimulation of the parasympathetic fiber of the frog glossopharyngeal nerve

When the glossopharyngeal nerve (GP) in the frog was strongly stimulated electrically, slow potentials were elicited from the tongue surface and taste cells in the fungiform papillae. Injection of atropine completely blocked these slow potentials. The present and previous data indicate that the slow potentials induced in the tongue surface and taste cells are due to a liquid junction potential between saliva secreted from the lingual glands due to parasympathetic fiber activity and an adapting solution on the tongue surface. Intracellularly recorded depolarizing receptor potentials in taste cells induced by 0.5 M NaCl and 3 mM acetic acid were enhanced by depolarizing slow potentials induced by GP nerve stimulation, but were depressed by the hyperpolarizing slow potentials. On average, the receptor potential of taste cells for 0.5 M NaCl was increased by 25% by the GP nerve-induced slow potential, but the receptor potential of taste cells for 3 mM acetic acid was decreased by 1% by the slow potential. These transformations of receptor potentials in frog taste cells were not due to a synaptic event initiated between taste cells and the efferent nerve fiber, but due to a non-synaptic event, a lingual junction potential generated in the dorsal lingual epithelium by GP nerve stimulation.     

Do Unique Proteins Exist in Taste Buds?

Proteins in papillae on the bovine tongue were analyzed by semi-micro, polyacrylamide gel electrophoresis. All the proteins in the papillae with taste buds were observed to be common to proteins in the surrounding epithelium without taste buds. The protein band which was reported to form a weak complex with compounds called sweet by man was also found in all parts of the tongue epithelium. The receptor molecules for chemical stimuli may be distributed in all the cells of the tongue epithelium or the content of receptor molecules in taste bud papillae may be extremely low.     

Biogenic Amines in the Vomeronasal Organ

The vomeronasal organ of frog and mouse was investigated forthe presence and content of serotonin and catecholamines bymeans of high-performance liquid chromatography. Measurableamounts of serotonin, adrenaline and noradrenaline were foundin the vomeronasal organ of adult individuals of both species.The amine content varied with sex of adult frogs and mice andsexual maturity of mice. In preliminary experiments, acute exposureto male urine containing pheromone affected the amine contentin the vomeronasal organ of adult female mice. These data suggestthat functional sex dimorphism is present in the vomeronasalorgan, and biochemical changes therein take place accordingto stage of sexual maturity. The role of biogenic amines inthe vomeronasal organ deserves further study. Chem. Senses 22:439–445, 1997.     

The effects of sialoadenectomy and exogenous EGF on taste bud morphology and maintenance

Taste buds on the dorsal tongue surface are continually bathedin saliva rich in epidermal growth factor (EGF). In the followingexperiment, taste bud number and morphology were monitored followingsubmandibular and sublingual salivary gland removal (sialoadenectomy),to determine if EGF plays a role in the maintenance and formationof taste buds. Adult male rats were divided into four groups:sialoadenectomized (SX, n = 4); sialoadenectomized with EGFreplacement (SX + EGF, n = 5); sham-operated (SH, n = 4); andsham-operated with exogenous EGF (SH + EGF, n = 5). After a3 week recovery, SX + EGF and SH + EGF animals were given 50µg/day EGF in their drinking water for 14 days. At day14, saliva was collected, the animals were killed and the presenceof EGF determined by radioligand-binding assay. Tongues wereremoved and histologically examined for the presence and morphologyof taste buds on fungiform and circumvallate papillae, or immunostainedfor the presence of EGF, TGF     

Biogenic amines in cultured neuroblastoma and astrocytoma cells

The presence of biogenic amines in cultured cells of mouse neuroblastoma C-1300 (clone NB-2a) was suggested by fluorescence-microscope histochemistry. Incubation in media containing L-[¹⁴C]tyrosine and L-[¹⁴C]tryptophan for 24 h, followed by high-voltage electrophoresis, radiochromatogram scanning, and scintillation counting, confirmed the presence of [¹⁴C]dopamine, [¹⁴C]norepinephrine, [¹⁴C]epinephrine, [¹⁴C]serotonin, [¹⁴C]tyramine, and [¹⁴C]octopamine. Dopamine, norepinephrine, epinephrine, and serotonin were demonstrated spectrophotofluorometrically in concentrations, expressed as micrograms amine per milligram protein, of 1.19, 0.027, 0.038, and 0.148, respectively, for cells in a stationary growth phase. Fluorescence-microscope histochemistry also suggested the presence of biogenic amines in cultured astrocytoma cells (cell line C6). Spectrophotofluorometric assay of cells in a stationary growth phase demonstrated intracellular dopamine, norepinephrine, epinephrine, and serotonin in concentrations significantly lower than those of neuroblastoma cells.     

Effect of larval density on whole-body biogenic amine levels of Tribolium freemani hinton

1. Various biogenic amines, their precursors and metabolites in gregarious and solitary forms of the flour beetle Tribolium freemani Hinton larvae were measured using high-performance liquid chromatography coupled with electrochemical detection.2. Octopamine was much more abundant than dopamine, serotonin, epinephrine, norepinephrine and related substances, suggesting that octopamine may play much more important roles in growth and behaviour of T. freemani than other biogenic amines and related substances.3. Octopamine levels were higher in gregarious forms than solitary forms of T. freemani larvae, and the relation between octopamine content, hormone cycles and motility is discussed.     

Inhibition of hemostasis by a high affinity biogenic amine-binding protein from the saliva of a blood-feeding insect

The saliva of the blood-feeding insect Rhodnius prolixus contains numerous pharmacologically active substances. Included among these are a number of lipocalin proteins that bind various ligands important in hemostasis and inflammation. One such protein is a biogenic amine-binding protein (ABP) that binds serotonin, epinephrine, and norepinephrine. Based on amino acid alignments, it is most similar to the nitrophorin group of lipocalins found in the same insect species. Physiologically, this protein appears to act as both a vasodilator and platelet aggregation inhibitor. This protein inhibits smooth muscle contraction of the rat uterus in response to serotonin and of the rabbit aorta in response to norepinephrine. Platelet aggregation induced by a combination of low concentrations of ADP and either serotonin or epinephrine is inhibited because of the binding of serotonin and epinephrine. Potentiation of aggregation induced by low concentrations of collagen along with serotonin or epinephrine is also inhibited. Dissociation constants for biogenic amines were measured using isothermal titration calorimetry and the Hummel-Dreyer method of equilibrium gel filtration. In this manner, K(d) values of 102, 24, and 345 nm were found for serotonin, norepinephrine, and epinephrine, respectively. Molecular modeling of ABP suggests that ligand binding is mediated by interaction with the side chains of aromatic amino acids and charged residues that line the binding pocket.     

Neural crest contribution to lingual mesenchyme, epithelium and developing taste papillae and taste buds

The epithelium of mammalian tongue hosts most of the taste buds that transduce gustatory stimuli into neural signals. In the field of taste biology, taste bud cells have been described as arising from "local epithelium", in distinction from many other receptor organs that are derived from neurogenic ectoderm including neural crest (NC). In fact, contribution of NC to both epithelium and mesenchyme in the developing tongue is not fully understood. In the present study we used two independent, well-characterized mouse lines, Wnt1-Cre and P0-Cre that express Cre recombinase in a NC-specific manner, in combination with two Cre reporter mouse lines, R26R and ZEG, and demonstrate a contribution of NC-derived cells to both tongue mesenchyme and epithelium including taste papillae and taste buds. In tongue mesenchyme, distribution of NC-derived cells is in close association with taste papillae. In tongue epithelium, labeled cells are observed in an initial scattered distribution and progress to a clustered pattern between papillae, and within papillae and early taste buds. This provides evidence for a contribution of NC to lingual epithelium. Together with previous reports for the origin of taste bud cells from local epithelium in postnatal mouse, we propose that NC cells migrate into and reside in the epithelium of the tongue primordium at an early embryonic stage, acquire epithelial cell phenotypes, and undergo cell proliferation and differentiation that is involved in the development of taste papillae and taste buds. Our findings lead to a new concept about derivation of taste bud cells that include a NC origin.     

Quantification of fungiform papillae and taste pores in living human subjects

A method developed to quantify taste buds in living human subjectsto study the relationship between taste sensitivity and tastebud distribution was used to count the taste buds in 10 humansubjects; fungiform papillae were mapped in 12 subjects. Tastebuds were identified by staining taste pores with methyleneblue, and images of the papillae and their taste pores wereobtained with videomicroscopy and an image processor. Fungiformpapillae showed a 3.3-fold range in density, from 22.1 to 73.6papillae/cm² with an average of 41.1 ± 16.8/cm² (s.d.,n = 2). There was a 14-fold range in taste pore density, from36 to 511 pores/cm² among subjects, with an average of 193 ±133/cm² (s.d., n = 10). Fungiform papillae contained from 0to 22 taste pores, with an average per subject of 3.75 ±1.4 taste pores/papilla (s.d., n = 10). We hypothesize thatsome differences in human taste sensitivity may be related tothese variations in taste bud density.     

The inverse relationship between the activity of pyridoxal kinase and the level of biogenic amines in rabbit brain

In a series of experiments, an inverse relationship was found to exist between the activity of pyridoxal kinase and the concentrations of brain norepinephrine, dopamine, and serotonin. When the biogenic amine content of the brain is lowered, the activity of pyridoxal kinase rises. Conversely, when the concentration of brain biogenic amines is elevated, a depression occurs in the activity of pyridoxal phosphokinase. This relationship, which resembles an in vivo allosteric inhibition phenomenon, not only is highly suggestive of a definite relation between the brain vitamin B₆ content and the metabolism of the biogenic amines, but also suggests that the concentration of brain biogenic amines might control the production of coenzyme pyridoxal phosphate.     

Is serotonin a chemical transmitter in the frog taste organ?

By artificially perfusing the frog tongue with serotonin (5HT) and its antagonists, the possibility of 5HT as a chemical transmitter from taste cells to nerve terminals in frog taste organ was examined. Although serotonin creatinine sulfate, when perfused through the lingual artery, produced impulse discharges in the glossopharyngeal nerve, creatinine sulfate elicited a similar response. Neural responses to taste stimuli were depressed by perfusion with 5HT. Among many antiserotonergic drugs perfused through the lingual artery, LSD was the only one which modified responses to taste stimuli. LSD suppressed taste responses to NaCl, CaCl₂ and water, while LSD at a high concentration (10^?5 g/ml) enhanced responses to guinine and HCl. When PCPA (DL-p-chlorophenylalanine) was injected intraperitoneally in conbination with reserpine, the agent did not significantly change taste responses. The above results possibly suggest that 5HT would not be a chemical mediator from taste cells to nerve terminals.     

Hormones in the nucleus. Immunologically demonstrable biogenic amines (serotonin, histamine) in the nucleus of rat peritoneal mast cells

Using 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide (EDAC) fixation and immunocytochemical confocal microscopic study, bright serotonin and histamine fluorescence appeared in the nucleus of rat peritoneal mast cells. In case of paraformaldehyde fixation, this was not observed. The phenomenon can be explained by the cross-linking effect of EDAC, which did not allow the efflux of biogenic amines from the nucleus. This means that biogenic amines are present in the nucleus of mast cells, and this is supported by the flow cytometric measurement data of the whole cell. Other hormones studied (triiodothyronine, insulin, and endorphin) were not present in the nucleus. Four pharmaca with biogenic amine-influencing character in the central nervous system were used for studying the relation between the external (surrounding and cytoplasmic) and nuclear biogenic amine content of mast cells. Fluoxetine, a serotonin reuptake inhibitor depleted nuclear as well as cytoplasmic serotonin content. Clorgyline, a MAO-A inhibitor, decreased cytoplasmic serotonin content and weakened nuclear serotonin fluorescence. The tryptophan hydroxylase inhibitor, para-chlorophenylalanine (PCPA), and the mast cell degranulator, Compound 48/80, reduced cytoplasmic serotonin content without influencing nuclear content. Histamine fluorescence was influenced solely by fluoxetine. The results show that nuclear 5-HT content is dependent firstly of serotonin uptake and reuptake. To our knowledge, this is the first exact report on the presence of non-steroid-type-receptor-transported hormones inside the nucleus of a cell.     

Secretin-cells of the mammalian intestine contain serotonin

Summary Various endocrine cells contain biogenic amines in addition to their peptide hormones. In the digestive tract, one of these amines is serotonin that is regularly present in enterochromaffin (EC-) cells. Previously, it has been assumed that other entero-endocrine cell types also contain this amine. Moreover, it was presumed that chromogranin A, an acidic glycoprotein, is involved in storage mechanisms for biogenic amines in endocrine cells. Using immunohistochemical techniques, we now exemplarily investigated cholecystokinin (CCK-) and secretin (S-) cells of five adult mammalian species for their content of serotonin and of chromogranin A. In all mammalian species, CCK-cells were devoid of serotonin but contained chromogranin A immunoreactivity of varying densities. In contrast, S-cells of all mammals were immunoreactive for serotonin; however, immunoreactivities for this biogenic monoamine were heterogeneous and varied from dense to faint or lacking immunostainings. Likewise, immunoreactivities for chromogranin A in S-cells showed inter-species and inter-cellular heterogeneities. S-cells containing serotonin were simultaneously immunoreactive for chromogranin A and the density of immunoreactivities for both were correlated in given S-cells. Based on mutual relationships of chromogranin A and serotonin immunoreactivities, we assume that chromogranin A is virtually a prerequisite for the S-cells' content of serotonin and that this protein participates in storage mechanisms for biogenic amines in endocrine cells.S-cells have now to be added to the family of amine-storing endocrine cells. Basically, serotonin-storing endocrine cells in the digestive tract cannot be simply regarded as enterochromaffin (EC-) cells any longer; the current nomenclature and classification of entero-endocrine cells should be reviewed in this respect.This work was supported by grants of the Deutsche Forschungs-gemeinschaft (EN 65/15-2)