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.     

A2BR adenosine receptor modulates sweet taste in circumvallate taste buds

In response to taste stimulation, taste buds release ATP, which activates ionotropic ATP receptors (P2X2/P2X3) on taste nerves as well as metabotropic (P2Y) purinergic receptors on taste bud cells. The action of the extracellular ATP is terminated by ectonucleotidases, ultimately generating adenosine, which itself can activate one or more G-protein coupled adenosine receptors: A1, A2A, A2B, and A3. Here we investigated the expression of adenosine receptors in mouse taste buds at both the nucleotide and protein expression levels. Of the adenosine receptors, only A2B receptor (A2BR) is expressed specifically in taste epithelia. Further, A2BR is expressed abundantly only in a subset of taste bud cells of posterior (circumvallate, foliate), but not anterior (fungiform, palate) taste fields in mice. Analysis of double-labeled tissue indicates that A2BR occurs on Type II taste bud cells that also express Gα14, which is present only in sweet-sensitive taste cells of the foliate and circumvallate papillae. Glossopharyngeal nerve recordings from A2BR knockout mice show significantly reduced responses to both sucrose and synthetic sweeteners, but normal responses to tastants representing other qualities. Thus, our study identified a novel regulator of sweet taste, the A2BR, which functions to potentiate sweet responses in posterior lingual taste fields.     

Ethanolamine Base-Exchange Reaction in Rat Brain Microsomal Subfractions

Crude microsomal fractions have been subfractionated by differential ultracentrifugation into subfractions A, B, and C, corresponding to light smooth, heavy smooth, and rough microsomal membranes, respectively. The purity and the vesiculation of the membranes were checked biochemically. Subfraction C showed the highest ethanolamine base-exchange activity, both on phospholipid and protein bases. The other two subfractions had roughly similar activities. The kinetic behavior of the enzyme activity, although anomalous, was similar in the three subfractions. Treatment of the vesicles with Pronase or with mercury-dextran produced inactivation of the ethanolamine base-exchange reaction in the three subfractions. These findings suggest that the active site of base-exchange activity would be localized on the external leaflet of the vesicles. Treatment of the membranes with trinitrobenzenesulfonic acid (TNBS) has shown that the newly synthesized phosphatidylethanolamine (PE) belongs to a pool easily reacting with the probe, independent of the subfraction investigated. On the other hand, the distribution of the bulk membrane PE reacting with TNBS differs in the three subfractions examined. It is concluded that the newly synthesized PE and probably the active site of the enzyme are on the external leaflet of the membrane in all subfractions and that the ethanolamine base-exchange reaction has similar properties in all subfractions.     

ISOLATION AND CHARACTERIZATION OF MYELIN-RELATED MEMBRANES

Abstract— Myelin related membrane fractions from rat brain and spinal cord were isolated from material normally discarded during standard myelin isolation procedures. A fraction which floated on 0.32 M-sucrose (F) and the material released after subjecting the myelin fraction to osmotic shock at two stages in the purification (W₁ and W₂) were characterized. These fractions were subjected to subfractionation on three step discontinuous sucrose gradients. Morphologically, the heavier subfrac-tions of W₁ and W₂ were shown to consist mainly of single membranes and vesicles. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis showed that, relative to myelin, proteolipid and basic protein were reduced in all subfractions, while the high molecular weight proteins were increased. The specific activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) was up to 2-fold higher than that of myelin in the heavier subfractions of W₁ and W₂. The major myelin-associated glycoprotein was also increased in these subfractions as determined by periodic acid-Schiff staining. Differential centrifugation of the initial tissue homogenate to remove microsomes prior to myelin isolation gave rise to W₁ and W₂ subfractions with a CNP specific activity 3–4 times that of myelin. The high molecular weight proteins and glycoproteins were enriched in these microsome-depleted subfractions, but were qualitatively similar to those of myelin. Some of the membranes in these fractions may be derived from the continuum between the plasma membrane of the oligodendrocyte and compact myelin. Fraction F consisted of small membrane fragments and many vesicles, and was particularly deficient in proteolipid. The specific activity of CNP in fraction F was about the same as myelin, while the major myelin associated glycoprotein could not be detected. Fraction F from normal CNS tissue appears to be similar to the floating fractions previously isolated in larger amounts from pathological brain undergoing edematous demyelination.     

FRACTIONATION OF OLFACTORY TISSUE HOMOGENATES. ISOLATION OF A CONCENTRATED PLASMA MEMBRANE FRACTION

Abstract— Differential and sucrose-density-gradient centrifugation techniques were used for studies on the separation of subcellular particles from rabbit brain and olfactory tissue. Comparisons were made among various fractions from the two types of tissue. These comparisons included protein concentration and enzyme activities of the individual fractions as well as their distribution in subfractions from density gradient separations. In tissue whole homogenates, the percentage of total ATPase activity as ouabain sensitive Na⁺-K⁺ ATPase activity was about 4 times greater in brain cortex (63 per cent) than in olfactory tissue (17 per cent). Cytochrome oxidase and Na⁺-K⁺ ATPase activities were used to indicate the presence and the concentration of mitochondria and of the plasma membranes. A fraction with properties similar to the mitochondria plus nerve ending fraction from brain homogenates (fraction B) was obtained from olfactory tissue. Nerve ending concentration subfractions (B₂) were prepared from the B primary fractions. Plasma membrane subfractions were obtained by osmotic shock treatment of B₂, In the fraction of plasma membrane from olfactory tissue (E₂), 56 per cent of the total ATPase activity was Na⁺-K⁺ ATPase activity. In E₂ from brain 71 per cent was Na⁺-K⁺ ATPase activity. Deoxycholate (DOC)-treated fractions containing nerve endings from brain preparations showed much greater increase in cytochrome oxidase activity than did similar fractions from olfactory tissue. DOC treatment increased the NADH cytochrome c reductase activity of all fractions and subfractions from brain, while it decreased activity in all but one fraction from olfactory tissue. DOC treatment decreased both the Mg²⁺ and Na⁺-K⁺ ATPase activities in both types of tissue. Electron photomicrographs of olfactory B₂, B₃, E₂ and E₃ show clear morphological differences among these subfractions. The presence of possible cilia and basal bodies on vesicles in B₂ gives morphological evidence for the presence of terminal swellings in this subtraction in agreement with enzyme marker activity results.     

Rabbit myocardial membrane Ca2+-adenosine triphosphatase activity: stimulation in vitro by thyroid hormone

The in vitro stimulation of human and rabbit erythrocyte membrane Ca2+-ATPase activity by physiological concentrations of thyroid hormone has recently been described. To extend these observations to a nucleated cell model, Ca2+-ATPase activity in a membrane preparation obtained from rabbit myocardium has been studied. Activity of 5'-nucleotidase in the preparation was increased 26-fold over that of myocardial homogenate, consistent with enrichment by sarcolemma. Mean basal enzyme activity in membranes from nine animals was 20.8 +/- 3.3 mumol Pi mg membrane protein-1 90 min-1, approximately 20-fold the activity described in rabbit red cell membranes. Exposure of heart membranes in vitro to L-thyroxine (T4) (10(-10)M) increased Ca2+-ATPase activity to 29.2 +/- 3.8 mumol Pi (P less than 0.001). Dose-response studies conducted with T4 showed that maximal stimulatory response was obtained at 10(-10) M). Hormonal stimulation was comparable for L-T4 and triiodo-L-thyronine (T3) (10(-10) M). Tetraiodothyroacetic acid was without biological activity, whereas triiodothyroacetic acid and D-T4, each at 10(-10) M, significantly decreased enzyme activity compared to control (basal) levels. The action of L-T4 on myocardial membrane Ca2+-ATPase activity was inhibited by trifluoperazine (100 microM) and the naphthalenesulfonamide W-7 (50-100 microM), compounds that block actions of calmodulin, the protein activator of membrane-associated Ca2+-ATPase. Radioimmunoassay revealed the presence of calmodulin (1.4 micrograms/mg membrane protein-1) in the myocardial membrane fraction and 0.35 micrograms/mg-1 in cytosol. Myocardial Ca2+-ATPase activity, apparently of sarcolemmal origin, is thus thyroid hormone stimulable. The hormonal responsiveness of this calcium pump-associated enzyme requires calmodulin.     

The distribution of A1 adenosine receptor and 5′-nucleotidase in pig brain cortex subcellular fractions

Pig brain cerebral cortex was subfractionated by isopycnic centrifugation in sucrose gradients. In each subfraction the content of the agonist [³H]R-PIA binding, the activity of adenosine metabolizing enzymes (5-nucleotidase and adenosine deaminase) and the activity of membrane marker enzymes were determined. The fractions were also examined by electron microscope. In general, the results suggest a widespread distribution of A₁ adenosine receptors in membranes from different origins. Marker enzyme profile characterization indicated an enrichment of A₁ adenosine receptor in pre-synaptic membranes isolated from the crude synaptosomal fraction (P2B subfraction) as well as in membranes of glial origin such as myelin. The receptor is also present in the endoplasmic reticulum and in membranes isolated from the microsomal fraction that seem to have a post-synaptic origin (P3B). In subfractions having a high content of adenosine receptor the equilibrium binding paramters were obtained as well as the proportion of high- to low-affinity sites. From the values of the equilibrium constants it was not possible to find differences between the receptor in the different subfractions. Analysis of the affinity state distribution showed a diminished percentage of high-affinity sites in fraction P3A, which can be accounted by the existence of myelin membranes; in contrast the percentage of high-affinity states was higher in P2 and P3B, indicating that in these fractions the receptor is present in synaptosomal membranes. The close correlation shown between the enzyme 5-nucleotidase specific activity and the specific ligand binding distributions led us to postulate an important role for the enzyme in the regulation of adenosine action in pig brain cortex.     

Insulin-Like Growth Factors Are Expressed in the Taste System,but Do Not Maintain Adult Taste Buds

Growth factors regulate cell growth and differentiation in many tissues. In the taste system, as yet unknown growth factors are produced by neurons to maintain taste buds. A number of growth factor receptors are expressed at greater levels in taste buds than in the surrounding epithelium and may be receptors for candidate factors involved in taste bud maintenance. We determined that the ligands of eight of these receptors were expressed in the E14.5 geniculate ganglion and that four of these ligands were expressed in the adult geniculate ganglion. Of these, the insulin-like growth factors (IGF1, IGF2) were expressed in the ganglion and their receptor, insulin-like growth factor receptor 1 (IGF1R), were expressed at the highest levels in taste buds. To determine whether IGF1R regulates taste bud number or structure, we conditionally eliminated IGF1R from the lingual epithelium of mice using the keratin 14 (K14) promoter (K14-Cre::Igf1r^lox/lox). While K14-Cre::Igf1r^lox/lox mice had significantly fewer taste buds at P30 compared with control mice (Igf1r^lox/lox), this difference was not observed by P80. IGF1R removal did not affect taste bud size or cell number, and the number of phospholipase C β2- (PLCβ2) and carbonic anhydrase 4- (Car4) positive taste receptor cells did not differ between genotypes. Taste buds at the back of the tongue fungiform taste field were larger and contained more cells than those at the tongue tip, and these differences were diminished in K14-Cre::Igf1r^lox/lox mice. The epithelium was thicker at the back versus the tip of the tongue, and this difference was also attenuated in K14-Cre::Igf1r^lox/lox mice. We conclude that, although IGFs are expressed at high levels in the taste system, they likely play little or no role in maintaining adult taste bud structure. IGFs have a potential role in establishing the initial number of taste buds, and there may be limits on epithelial thickness in the absence of IGF1R signaling.     

Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate

Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) P0-Cre/R26-tdTomato (RFP) to label NC, NC derived Schwann cells and derivatives; (2) Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3) Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC.     

Morphometry and cellular dynamics of denervated fungiform taste buds in the hamster

For most species and gustatory papillae denervation resultsin a virtual disappearance of taste buds. This is not the casefor hamster fungiform papillae, which contain taste buds thatsurvive denervation. To characterize these taste buds, in thisstudy, counts and measurements were made of all buds on theanterior 3 mm of the hamster tongue at 36 or 91 days after resectingthe chorda/lingual nerve. Taste bud numbers were, at both timeperiods, unaffected by denervation. However, bud dimensionswere affected with denervated buds 25–30% smaller thancontrol ones. Counts of taste bud cells indicated that decreasesin bud size may result from shrinkage, but not a loss of cells.Tritiated thymidine autoradiography was used to evaluate whetherdenervation influences the mitotic activity or the migratorypattern of bud cells. For every animal, the average number oflabelled cells per bud was slightly lower on the denervatedthan the control side of the tongue. However, when labelledcell positions were evaluated at 0.25, 3 and 6 days after thymidine,the distances from the sides of the bud increased at increasingtimes after injection for both the innervated and the denervatedbuds. Stem cells were located laterally or basally in the bud.Labelled cells that migrated into the centers of the buds werefew and seen only at 6 days post-injection time in both controland experimental buds. The moderate effects of denervation ontaste bud sizes and mitotic activities may indicate a generalizedatrophy. Remarkably intact were taste bud numbers and the migratorypatterns of cells, features of anterior tongue taste buds inthe hamster that are relatively invulnerable to resection ofthe chorda /lingual nerve.     

Neuron/target matching between chorda tympani neurons and taste buds during postnatal rat development

During postnatal development, a relationship is established between the size of individual taste buds and number of innervating neurons. To determine whether rearrangement of neurons that innervate taste buds establishes this relationship, we labeled single taste buds at postnatal day 10 (P10) and again at either P15, P20, or P40 with retrograde fluorescent neuronal tracers. The number of single- and double-labeled geniculate ganglion cells was counted, and the respective taste bud volumes were measured for the three groups of rats. The current study replicates findings from an earlier report demonstrating that the larger the taste bud, the more geniculate ganglion cells that innervate it. This relationship between taste bud size and number of innervating neurons is not apparent until P40, when taste bud size reaches maturity. These findings are extended here by demonstrating that the number of neurons that innervate taste buds at P10, when taste bud size is small and relatively homogeneous, predicts the size that the respective taste bud will become at maturity. Moreover, while there is some neural rearrangement of taste bud innervation from P10 to P40, rearrangement does not impact the relationship between taste bud size and innervating neurons. That is, the neurons that maintain contact with taste buds from P10 through P40 accurately predict the mature taste bud size. Therefore, the size of the mature taste bud is determined by P10 and relates to the number of sensory neurons that innervate it at that age and the number of neurons that maintain contact with it throughout the first 40 days of postnatal development.     

Changes in Parameters of the Plasmalemma ATPase during Peach Vegetative Bud Dormancy

Plant dormancy and dormancy breaking depend, at least partially, on close relationships between buds and tissues underlying bud (bud stands). In Prunus persica, the dormancy was related to high nutrient absorption in bud stands linked to high plasmalemma ATPase (EC 3.6.1.3) activity. Two plasmalemma fractions was isolated from peach vegetative buds and bud stands using aqueous phase partitioning and ultracentrifugation. Results of markers enzyme assays indicated that both plasmalemma enriched fractions obtained were highly purified. During the dormancy period plasma membrane ATPase amount and activity were higher in bud stands than in buds. Moreover, assays performed at different temperatures (4, 18, 30 °C) indicated modifications of kinetic parameters (K_m, V_m) in both tissues during dormancy release. In buds, from November to February, K_m declined at 4°C and increased at 30 °C whereas no changes was measured at 18 °C and V_m increased at all temperature. In bud stands, no changes of K_m was measured at 4 °C and 18 °C whereas an increase occurred at 30 °C and V_m decreased at all temperature. According to the results, it can be postulated that dormancy release in peach-tree could be related to modifications of plasma membrane ATPase properties, in buds and bud stands, during winter time.     

Isolation of Golgi fractions from colchicine-treated rat liver. II. Electrophoretic characterization.

1. Intact Golgi fractions, three from colchicine- or ethanol-treated rat livers and two from a control, were analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. All the fractions showed very similar electrophoretic profiles with 33 protein bands, some of which, especially albumin, had rather higher density in the secretory vesicle fraction than those in the cisternal fraction. 2. Using albumin as the content marker, the Golgi fractions were subfractionated into membranes and contents by freezing-thawing and sonication followed by centrifugation. Distribution of galactosyltransferase among these membrane preparations showed that this enzyme was more enriched in the Golgi cisternal membranes than in the secretory vesicle membranes. 3. All the membrane preparations from the Golgi complex showed very similar patterns on electrophoresis, which were distinctly different from those of microsomal membranes and of plasma membrane. Furthermore, all the Golgi content subfractions had similar protein components, most of which were also found in serum. The microsomal contents, however, showed a considerably different pattern from those of the Golgi contents. 4. From these results it could be concluded that the secretory vesicles are indeed a member of the Golgi complex despite their different appearance and morphology.     

Changes in number and morphology of fungiform taste buds in rat after transection of the chorda tympani or chordalingual nerve

In normal rats there is one taste bud on the apical surfaceof each fungiform papilla. These taste buds are innervated bythe chorda tympani proper nerve (CT). According to general consensus,after cutting the nerve the taste buds should disappear. Inthis study, performed on 24 rats divided in six groups, theCT nerve on the left side (singly denervated) and the combinedchorda-lingual (CT-L) nerve on the other side (doubly denervatedwere permanently interrupted. The animals were sacrificed after5, 10, 20, 35,60 and 100 days and their tongues serially sectionedfor light microscope examiation. Some papillae were examinedunder an electron microscope. The papillae were categorizedinto three groups: papillae with a normal looking taste bud,with an abnormal looking taste bud and without a taste bud.The results showed a substantial number of papillae with a normallooking taste bud present at all time intervals in all animals.More specifically, on the singly denervated side the proportionof normal looking taste buds stayed below 10% until day 60,when it increased to 15% and to 23% on day 100. The proportionof abnormal looking taste buds decreased from above 92% by day5 to 49% on day 100. The percentage of fungiform papillae withoutsigns of a taste bud was relatively low on the singly denervatedside at times (1, 5, 16, 29, 34 and 28%). On the doubly denervatedside fewer than than 4% normal looking taste buds were foundthroughout the time period. The proportion of abnormal lookingtaste buds decreased from {small tilde} 96% by day 5 to 35%on day 100. A significantly higher proportion of papillae withno taste bud was observed on this side from day 10 onwards.(1, 29, 32, 52, 60 and 63%). The reasons for the differencein tast bud number between the doubly and singly denervatedsides are unknown, but it is possible that collaterals fromother (non-gustatory) nerves have an ability, although limited,to induce and maintain fungiform taste buds. In other words,the capacity to induce taste bud formation is not limited exclusivelyto gustatory nerves.     

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     

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.     

Taste bud density in circumvallate and fungiform papillae of the bovine tongue

Taste bud quantitation may provide useful parameters for interspecies comparisons of the gustatory system. The present study is a morphometric analysis of bovine taste papillae. Circumvallate and fungiform papillae from six bovine tongues were serially sectioned and, following staining, analyzed. Circumvallate papillae were found to have a mean volume of 3.66 +/- 2.82 mm3, a mean number of taste buds per papilla of 445 +/- 279, and a mean taste bud density of 155 +/- 112 buds/mm3. Values for lateral fungiform papillae for the same three parameters were 0.384 +/- 0.184 mm3, 13.2 +/- 13.4, and 40.8 +/- 46.6 buds/mm3, respectively. Values for dorsal fungiform papillae were 0.438 +/- 0.246 mm3, 4.39 +/- 4.78, and 14.0 +/- 17.1 buds/mm3, respectively. Circumvallate papillae were found to have a significantly greater volume, number of taste buds per papilla, and taste bud density than either type of fungiform papilla. These data should serve as background for biochemical, endocrinological, or neurological studies involving the bovine tongue.