The fine structural effect of sialectomy on the taste bud cells in the rat.

Taste buds in the rat and other mammals share a secretory activity with their transduction function as taste receptor. The present work shows the effect of bilateral removal of the main salivary glands on taste bud cells' components related to secretion in the vallate papilla of the rat. In the sialectomized rats remarkable changes were evidence in the dark and intermediate types of taste bud cells, which are known to be the secretory components. Such changes involve hypertrophy of either the protein synthetizing machinery, the smooth endoplasmic reticulum or the Golgi complex. Lucent and coated vesicles associated to Golgi cisternae increased in number but the amount of dense-core vesicles (secretory vesicles) at the apical cytoplasm of cells decreased. Images of exocytosis of secretory products were observed. The hypertrophy of Golgi complex components was clearly detected with the OsO4 impregnation method for light and electron microscopy. Alteration in the acid phosphatase activity of taste bud cells was not observed in the sialectomized rats. These findings suggest that sialectomy stimulates the entire secretory cycle of dark and intermediate taste bud cells. The light taste bud cells, which are not engaged in secretion, are hardly affected by the treatment. Although taste buds in mammals are neuro-dependent structures, present evidence indicates that they are also sensitive to non-neural influences.     

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     

Ecto-calcium-dependent ATPase activity of mammalian taste bud cells.

Histochemistry was utilized to characterize Ca-ATPases associated with lingual taste buds in the golden hamster. Taste buds showed elevated staining for magnesium- or calcium-dependent ATPase (Ca-ATPase) relative to the surrounding epithelium. At low calcium concentrations (0.1-0.5 mM), intracellular staining predominated. Most of the studies were conducted at calcium concentrations of > or = 10 mM, in which most of the staining was localized to the external face of plasma membranes of taste bud cells (including receptor and basal cells) located in the core of fungiform taste buds, or the entire vallate or foliate taste buds. The peripheral fungiform taste bud cells stained much less intensely, but the peripheral cells adjacent to the core showed intermediate levels. GTP and ITP were just as effective substrates as ATP. Millimolar concentrations of magnesium were as effective as calcium. Inhibitors of intracellular ATPases, including quercetin, sodium azide, and 2,4-dinitrophenol, had no effect on the staining. Therefore, the Ca-ATPase staining of plasma membranes at mM concentrations of calcium is thought to correspond to one or more ecto-Ca-ATPase activities with unknown functions. Roles related to increased energy requirements or to the possible function of ATP as a neurotransmitter or -modulator are proposed.     

Apical secretion from taste bud and other epithelial cells in amphibians

Summary Taste buds of the axolotl, Ambystoma mexicanum, contain cells, previously undescribed in this species, which have a long apical process, and are similar to the Type III cells of mammalian taste buds, and to the gustatory cells in fish. In the supporting cells, there is evidence of periodic decapitation, in addition to secretion by exocytosis. Bilaminar fragments, which are leafshaped bodies formed of two dense laminae separated by a lucent gap, protrude from the apical microvilli of the supporting cells and are found detached in the extracellular secreted layer. Their form and dimensions suggest that they represent secreted lipo-protein material. Similar bilaminar fragments have been seen, in much smaller numbers, on some other epithelial cells in amphibians, and in fish. A unique case, in which rough endoplasmic reticulum was found in the extracellular layer of the axolotl oral epithelium, is reported; it had apparently been ejected from the cell. It is suggested that the axolotl produces a copious secretion at the taste bud pore, in order to wash the surface, and that the bilaminar fragments represent material aiding this cleansing process. The condition in the axolotl is compared with that in some other species, particularly Rana temporaria.The author wishes to thank Professor E.G. Gray, F.R.S., for the use of a tilting stage, and Mr. E. Perry for technical assistance     

Zinc localization in taste bud membranes

Zinc was measured by flame aspiration atomic absorption spectrophotometry in homogenates and in enriched fractions and subfractions from bovine taste bud membranes and from surrounding control tissues that contained no taste buds. Zinc was found in significantly higher concentrations in tissues containing taste buds and increased in concentration as biochemical and electron microscopic purity increased. The role of zinc in taste bud membranes could relate to its role in membrane stabilization or to its activity in alkaline phosphatase, a zinc-dependent enzyme whose specific activity increased in taste bud membranes in the same manner as did zinc concentration.     

Intracellular characteristics and responses of taste bud and lingual cells of the mudpuppy

Intracellular recordings of membrane potentials of mudpuppy lingual cells were made with micropipette electrodes. Three types of cells were distinguished by their responses to chemical stimulation. Surface epithelial (SE) cells outside of taste buds responded with large membrane potential and resistance changes to a variety of stimuli representing the four taste qualities. Salts and acids evoked particularly large potential changes, and MgCl2, acids, and quinine greatly increased the membrane resistance. One type of taste bud cell (TB-1) was characterized by large depolarizations to K salts, and the other type of taste bud cell (TB-2) characteristically hyperpolarized to MgCl2, acid, and sugar solutions. Membrane resistance changes accompanying TB-1 and TB-2 cell responses were relatively small compared to those of SE cells. Electrotonic coupling was observed between pairs of SE and TB-2 cells but not for pairs of TB-1 cells nor cells of different types. After recording cell responses, dye-marking allowed verification of results in situ and histologically. From the identification of cells in section, it is hypothesized the TB-1 and TB- 2 cells correspond to light and dark cells, respectively. Responses of TB-1 cells imply a taste receptive function; wheras TB 2-cell responses suggest secretory, supportive, and (or) receptive functions. Factors affecting cellular characteristics, non-taste bud cell responsiveness, response mechanisms, and function of electrotonic coupling are discussed in relation to taste reception.     

The effect of temperature on post-feeding ammonia excretion and oxygen consumption in the southern catfish

The post-prandial rates of ammonia excretion (TAN) and oxygen consumption in the southern catfish (Silurus meridionalis) were assessed at 2 h intervals post-feeding until the rates returned to those of the fasting rates, at 17.5, 22.5, 27.5, and 32.5°C, respectively. Both fasting TAN and increased with temperature, and were lower than those previously reported for many fish species. The relationship between fasting TAN (mmol NH₃–N kg⁻¹ h⁻¹) and temperature (T, °C) was described as: fasting TAN = 0.144e ^0.0266T (r = 0.526, n = 27, P < 0.05). The magnitude of ammonia excretion and its ratio to total N intake during the specific dynamic action (SDA) tended to increase initially, and then decrease with increasing temperature. The ammonia quotient (AQ), calculated as mol NH₃–N/mol O₂, following feeding decreased as temperature increased. The relationship between AQ during SDA and temperature was described as: AQ_{during SDA} = 0.303e ^−0.0143T (r = 0.739, n = 21, P < 0.05). Our results suggest that ammonia excretion and oxygen consumption post-feeding are operating independently of each other. Furthermore, it appears that the importance of protein as a metabolic substrate in postprandial fish decreases with temperature.     

Glutamate-induced cobalt uptake elicited by kainate receptors in rat taste bud cells

Glutamate-induced cobalt uptake reveals non-N-methyl-D-aspartate (non-NMDA) glutamate receptors (GluRs) in rat taste bud cells. However, it is not known which type of non-NMDA glutamate receptors is involved. We used a cobalt staining technique combined with pharmacological tests for kainate or alpha-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptors and/or immunohistochemistry against subunits of GluRs to examine the presence of non-NMDA receptors in rat foliate tastebud cells. Cobalt uptake into taste cells was elicited by treating taste buds with glutamate, kainate or SYM 2081, a kainate receptor agonist. Treating taste buds with AMPA or fluorowillardiine did not stimulate significant cobalt uptake. Moreover, 6-cyano-7-nitro-quinoxaline-2, 3-dione significantly reduced cobalt staining elicited by glutamate or kainate receptor agonists, but SYM 2206, an AMPA receptor antagonist, did not. Immunohistochemistry against subunits of GluRs reveals GluR6 and KA1-like immunoreactivity. Moreover, most glutamate-induced cobalt-stained cells showed GluR6 and KA1-like immunoreactivity. These results suggest that glutamate-induced cobalt uptake in taste cells occurs mainly via kainate type GluRs.     

Biochemical studies of taste sensation. III. Preparation of a suspension of bovine taste bud cells and their labeling with a fluorescent probe.

A method to prepare suspensions of taste bud cells is described. Bovine circumvallate papillae, which contain most of the taste buds in this animal, are incubated in collagenase-containing medium and the epidermal sidewall tissue is then dissected from the inner gelatinous dermis. The sidewall tissue, which contains the taste buds, is gently homogenized by manual operation of an all-glass homogenizer with a loose-fitting pestle. The suspended material is separated on a discontinous Ficoll gradient (2%, 8%, 10%, 12% w/w). The material banding at the 8-2% interface is greatly enriched in spindle-shaped cells that are morphologically similar to taste bud cells as they appear in situ. These cells are not seen when the procedure is done with tissues devoid of taste buds, namely the upper surface of the circumvallate papilla or epithelium from the intermolar eminence. Fluorescence analysis indicates that the hydrophobic probe, 8-anilino-1-naphthalenesulfonate (ANS), binds to relatively nonpolar sites in the suspension. It is postulated that the probe is adsorbing onto the surface membrane of the cell. These preparations may be useful in studying specificity and transduction in taste sensation.     

Immunocytochemistry of glutathione S-transferase in taste bud cells of rat circumvallate and foliate papillae

Immunocytochemistry was used to investigate the distribution of cells reacting with specific antibodies against glutathione S-transferase (GST) mu and pi in rat circumvallate and foliate taste buds; the findings were confirmed by Western blotting. Double immunofluorescence staining for protein gene product (PGP) 9.5 and GST subunits allowed the classification of taste bud cells of both papillae into: (i) cells immunoreactive to either PGP 9.5 or GST subunit antibody; (ii) cells immunoreactive to both antibodies; and (iii) cells that did not react with either of these antibodies. Immunoelectron microscopy revealed that most GST subunit-immunoreactive cells seemed to be either type II or type III cells based on their ultrastructure. Since PGP 9.5 is now widely used as a marker for type III cells in mammalian taste buds, it seems reasonable to believe that most GST subunit-immunoreactive cells are type II cells. Whether cells immunoreactive for both PGP 9.5 and GST subunits constitute a small subpopulation of type III cells or whether they are intermediate forms between type II and III cells is under investigation. No type I cells reacted with antibodies against GST subunits in the present study. GST subunits in taste bud cells may participate in xenobiotic metabolism of certain substances exposed to taste pits, as already shown for olfactory epithelium.     

Biochemical studies of taste sensation. III. Preparation of a suspension of bovine taste bud cells and their labeling with a fluorescent probe

A method to prepare suspensions of taste bud cells is described. Bovine circumvallate papillae, which contain most of the taste buds in this animal, are incubated in collagenase-containing medium and the epidermal sidewall tissue is then dissected from the inner gelatinous dermis. The sidewall tissue, which contains the taste buds, is gently homogenized by manual operation of an all-glass homogenizer with a loose-fitting pestle. The suspended material is separated on a discontinuous Ficoll gradient (2%, 8%, 10%, 12% w/w). The material banding at the 8–2% interface is greatly enriched in spindle-shaped cells that are morphologically similar to taste bud cells as they appear in situ. These cells are not seen when the procedure is done with tissues devoid of taste buds, namely the upper surface of the circumvallate papilla or epithelium from the intermolar eminence. Fluorescence analysis indicates that the hydrophobic probe, 8-anilino-1-naphthalenesulfonate (ANS), binds to relatively nonpolar sites in the suspension. It is postulated that the probe is adsorbing onto the surface membrane of the cell. These preparations may be useful in studying specificity and transduction in taste sensation.     

Distribution of vimentin in the developing chick taste bud during the perihatching period.

The tissue environment within which taste bud cells develop has not been wholly elaborated. Previous studies of taste bud development in vertebrates, including the avian chick, have suggested that taste bud cells could arise from one, or several tissue sources (e.g. crest-mesenchyme, local ectoderm or endoderm). Thus, molecular markers which are present in gemmal as well as interfacing (peribud epithelium; mesenchyme-epithelium) regions, and their degree of expression during stages of taste bud development, are of special interest. The intermediate filament protein, vimentin, occurs in mesenchymal and mesodermally-derived (e.g. endothelial, fibroblast) cells as well as highly proliferating epithelium (e.g. tumors). The present study in chick gustatory tissue utilized antibodies against vimentin and the avidin-biotin-peroxidase technique to evaluate vimentin immunoreactivity (IR) within a timeframe which includes: 1) early stages of the taste bud primordium [embryonic days (E)17-E18)]; 2) the beginning of an accelerated bud cell proliferation at the time of initial, taste bud pore opening [around E19]; 3) attaining the adult complement of taste buds [around posthatch (H) day 1], and 4) completed organogenesis (H 17). During this time span, vimentin-IR was characterized in a region including and sometimes bridging taste bud and subepithelial connective tissue, whereas non-gustatory surrounding epithelium and salivary glands were vimentin-immuno-negative. Intragemmally, the proportion of vimentin-IR cells as related to total taste bud cells peaked at E19. These results indicate that vimentin expression, in part, is related to the onset of taste bud cell proliferation and suggest that mesenchyme could be one source of taste bud cells. Secondly, fibronectin, an extracellular matrix component of the epithelial basement membrane interface with mesenchyme, was expressed at or near the apical surfaces of taste bud cells projecting into the bud lumen, and in the basal gemmal region suggesting the possible role of fibronectin as a chemotactic anchor for differentiating and migrating taste bud receptor cells. Lastly, neuron-specific enolase-IR indicates that axonal varicosities are already present intragemmally at E17-E18, that is, during the incipient period of identifiable taste bud primordia.     

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.     

Sucrose-stimulated subsecond transient increase in cGMP level in rat intact circumvallate taste bud cells

Initial sweet tastetransduction is expected to occur in the subsecond time range. Wedemonstrate a rapid and transient (75-250 ms) increase of cGMP(but not cAMP) level in rat intact circumvallate taste cells afterstimulation by sucrose. This rapid increase does not occur innonsensory epithelial cells. Pretreatment with a nonspecificphosphodiesterase (PDE) inhibitor (IBMX), a specific cAMP-PDE4inhibitor (denbufylline), or an adenylyl cyclase activator (forskolin)all increased basal cAMP and abolished the sucrose-stimulated cGMPincrease at 150 ms. Pretreatment with a soluble guanylyl cyclaseinhibitor(1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) reduced, whereas a specific cGMP-PDE inhibitor (zaprinast) abolished, the sucrose-stimulated cGMP increase. It is proposed that cGMP isinvolved in the initial stage of sugar taste transduction and that cGMPis more significant than cAMP at this stage. Activation of solubleguanylyl cyclase and inhibition of cGMP-PDE may be involved in thetransient elevation of cGMP in response to sucrose stimulation.Moreover, it appears that cAMP level must remain low for sucrose tostimulate an increase in cGMP.