Identification of 5'-adenylylimidodiphosphate-hydrolyzing enzyme activity in rabbit taste bud cells using X-ray microanalysis

X-ray microanalysis has been used to characterize the enzyme activity hydrolyzing the ATP analogue 5'-adenylylimidodiphosphate (AMP-PNP) in taste bud cells. Rabbit foliate papillae fixed with paraformaldehyde and glutaraldehyde were incubated cytochemically with AMP-PNP as the substrate and lead ion as capture agent. The reaction product which appeared on the microvilli of taste bud cells was examined using an energy dispersive X-ray microanalyzer connected to an analytical electron microscope. The X-ray spectrum thus obtained was compared with that obtained from the product obtained from the demonstration of ATPase activity. Comparison of the phosphorus/lead ratios in the two products showed that twice as much phosphorus was released from an AMP-PNP molecule by the activity in question compared with that released from an ATP molecule by ATPase activity. This indicates that the enzyme hydrolyzes AMP-PNP into AMP and imidodiphosphate and that the enzyme is adenylate cyclase or ATP pyrophosphohydrolase, which possesses a similar hydrolytic property, but not ATPase or alkaline phosphatase, which hydrolyzes AMP-PNP into ADP-NH2 and orthophosphate. This paper provides an example of the use of X-ray microanalysis as a tool for enzyme distinction. The method is applicable to a variety of enzymes and tissues.     

Na K ATPase activity in mammalian erythrocytes

The effects of membranotropic substances--nonionic detergent Tween-20 and EDTA--on the activity and some properties of Na,K-ATPase from mammalian erythrocytes were studied. It was shown that pretreatment of whole erythrocytes with Tween-20 (5 mg/ml) allows a detection of the enzyme activity, which cannot be detected in intact cells. It was also found that erythrocyte ghosts with a high and stable activity of Na,K-ATPase can be obtained by injections of EDTA (1-2 mM) into the hemolysis medium. Although the enzyme activity in whole erythrocytes and their ghosts was detected by the use of various membranotropic agents, the type of the dependence of the Na,K-ATPase activity on MgCl2 and EDTA concentration in the incubation medium was essentially the same for both cell preparations, the optimal concentrations of MgCl2 and EDTA being 3 and 1 mM, respectively. A rise in MgCl2 concentration above 3 mM caused a decrease of the enzymatic activity. Simple techniques have been developed for the detection of the Na,K-ATPase activity in mammalian erythrocytes which allow the determination of a higher enzymatic activity than those described in literature.     

The effect of temperature on the turnover of taste bud cells in catfish.

Renewal of taste bud cells on the barbels of channel catfish was studied. Groups of catfish, held in and acclimitized to 14 degrees C, 18 degrees C, 22 degrees C and 30 degrees C dechlorinated tap water were injected with [3H]thymidine (3.0 muCi/g body weight intraperitoneally). Barbels were sampled at various times after injection and prepared for light microscope autoradiography. Results show that epithelial cells surrounding the taste buds divide and some of their daughter cells migrate into the taste buds. The time at which 50% of the labelled cells have degenerated is taken as the average turnover time or average life span of the taste bud cells. The average life span as well as the time spent inside the taste buds is highly temperature-dependent. At 14 degrees C, 18 degrees C, 22 degrees C and 30 degrees C the average life span is on the order of 40, 30, 15 and 12 days respectively. Further studies indicate that both light and dark staining cells of the taste bud were labelled.     

Sodium + potassium-activated ATPase of mammalian brain. Regulation of phosphatase activity.

1. The K+-nitrophenylphosphatase activity associated with mammalian brain (Na+ + K+)-ATPase displays K+ activation curves that have intermediary plateaus and maxima in the presence of less than saturating concentrations of Na+. Zero Na+ and saturating Na+ produce sigmoid K+-activation curves with low and high K+ affinities respectively. 2. ATP inhibits K+-activated nitrophenylphosphatase through both competitive and non-competitive mechanisms. ATP is synergistic with Na+ in the mechanism which converts the enzyme from low to high K+ affinity. 3. The Na+ and K+ interactions can be accounted for by equations which describe a model with separate regulatory sites for Na+ and K+ and with K+- requiring catalytic site which is only accessible in one of the two principal conformational stages of the enzyme. 4. The effects of ATP can be accounted for by the same model through interactions at a single nucleotide binding site. Inhibition which is competitive with K+ and non-competitive with substrate arises from stabilization of the inactive enzyme conformation. Inhibition which is non-competitive with K+ and competitive with substrate results from interactions with the active enzyme conformation. The synergism between Na+ and ATP appears to arise as a consequence of the formation of phosphoryl enzyme. 5. A model for (Na+ + K+)-ATPase is discussed which involves in-phase coupling of subunit interactions as suggested by these studies.     

Identification of an RNA-dependent ATPase activity in mammalian U5 snRNPs.

Nuclear pre-mRNA splicing requires ATP at several steps from spliceosome assembly to product release. Here, we demonstrate that an integral component of the 20S U5 snRNP is an RNA-dependent ATPase. The ATPase activity of 20S U5 and 25S [U4/U6.U5] snRNPs purified by glycerol gradient centrifugation is strongly stimulated by homopolymeric RNA but not ssDNA. Purified 12S Ul and U2 snRNPs do not exhibit ATPase activity. Moreover, the U5-associated NTPase specifically hydrolyzes ATP and dATP. The additional purification of 20S U5 snRNPs by Mono Q chromatography does not affect the efficiency of ATP hydrolysis. Both U5 and tri-snRNPs bind ATP stoichiometrically in an RNA-independent manner. A candidate ATPase was identified by UV-irradiation of purified snRNPs with radiolabeled ATP. In the presence of homopolymeric RNA, the 200 kDa U5-specific protein is the major crosslinked protein, even in Mono Q-purified U5 snRNPs. The correlation between RNA-dependent ATPase activity in the U5 snRNP and the RNA-dependent onset of this crosslink strongly suggests that the 200 kDa protein is an RNA-dependent ATPase. Furthermore, both the formation of the crosslink and ATPase activity appear with a similar substrate specificity for ATP.     

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     

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.     

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.     

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.     

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.     

Afferent neurotransmission mediated by hemichannels in mammalian taste cells

In mammalian taste buds, ionotropic P2X receptors operate in gustatory nerve endings to mediate afferent inputs. Thus, ATP secretion represents a key aspect of taste transduction. Here, we characterized individual vallate taste cells electrophysiologically and assayed their secretion of ATP with a biosensor. Among electrophysiologically distinguishable taste cells, a population was found that released ATP in a manner that was Ca(2+) independent but voltage-dependent. Data from physiological and pharmacological experiments suggested that ATP was released from taste cells via specific channels, likely to be connexin or pannexin hemichannels. A small fraction of ATP-secreting taste cells responded to bitter compounds, indicating that they express taste receptors, their G-protein-coupled and downstream transduction elements. Single cell RT-PCR revealed that ATP-secreting taste cells expressed gustducin, TRPM5, PLCbeta2, multiple connexins and pannexin 1. Altogether, our data indicate that tastant-responsive taste cells release the neurotransmitter ATP via a non-exocytotic mechanism dependent upon the generation of an action potential.     

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.     

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.     

Separation of large mammalian ventricular myosin differing in ATPase activity

To investigate a possible heterogeneity of human ventricular myosin, papillary muscles of patients with valvular dysfunction were examined using a modified native gel electrophoresis. Myosin was separated into 2 components termed VA and VB, whereby the VA to VB proportion appeared to depend on the ventricular load. The proportion of the faster migrating band VA was correlated (P<0.05) with end-diastolic pressure and the aortic pressure-cardiac index product. The regression based on these variables accounted for 67% of the variation in VA (R2=0.67). The VA proportion was, however, not significantly correlated with cardiac norepinephrine concentration. The ATPase activity of the 2 components of myosin was assessed from the Ca3(PO4)2 precipitation by incubating the gel in the presence of ATP and CaCl2. The ATPase activity of VA was 60% of that of VB. The VA and VB forms were observed also in the cat (31.4% VA), dog (32.1% VA), pig (28.5% VA), wild pig (33.7% VA), and roe deer (30.5% VA). VA and VB were not detected in the rat exhibiting the 3 isoforms V1, V2, and V3, rabbit (100% V3), and hare (86% V1). The data demonstrate a heterogeneity of large mammalian ventricular myosin, whereby an increased cardiac load appeared to be associated with a higher myosin VA proportion that exhibited a reduced ATPase activity.     

Fgf signaling controls pharyngeal taste bud formation through miR-200 and Delta-Notch activity

Taste buds, the taste sensory organs, are conserved in vertebrates and composed of distinct cell types, including taste receptor, basal/presynaptic and support cells. Here, we characterize zebrafish taste bud development and show that compromised Fgf signaling in the larva results in taste bud reduction and disorganization. We determine that Fgf activity is required within pharyngeal endoderm for formation of Calb2b(+) cells and reveal miR-200 and Delta-Notch signaling as key factors in this process. miR-200 knock down shows that miR-200 activity is required for taste bud formation and in particular for Calb2b(+) cell formation. Compromised delta activity in mib(-/-) dramatically reduces the number of Calb2b(+) cells and increases the number of 5HT(+) cells. Conversely, larvae with increased Notch activity and ascl1a(-/-) mutants are devoid of 5HT(+) cells, but have maintained and increased Calb2b(+) cells, respectively. These results show that Delta-Notch signaling is required for intact taste bud organ formation. Consistent with this, Notch activity restores Calb2b(+) cell formation in pharyngeal endoderm with compromised Fgf signaling, but fails to restore the formation of these cells after miR-200 knock down. Altogether, this study provides genetic evidence that supports a novel model where Fgf regulates Delta-Notch signaling, and subsequently miR-200 activity, in order to promote taste bud cell type differentiation.