Occurrence of gustducin-immunoreactive cells in von Ebner’s glands of guinea pigs

An immunohistochemical examination of guinea-pig taste buds in vallate papillae revealed gustducin-immunoreactive cells in the area of von Ebner’s glands, minor salivary glands. Since there have been no reports describing those cells in these locations for other species, we investigated these glands in order both to localize the cells and compare their immunoreactive characteristics with corresponding cells in the vallate taste buds. The gustducin-immunoreactive cells coincided with cells containing no secretory granules in the end portion of the glands, which was supported by the electron-microscopic immunocytochemistry. Double immunofluorescence microscopy confirmed these cells to be entirely immunopositive to type III inositol 1,4,5-triphosphate receptor (IP₃R-3), phospholipase Cβ2 (PLCβ2), and villin and also partly immunopositive to neuron-specific enolase (NSE) and calbindin D-28K. The gustducin-immunoreactive cells in the vallate taste buds exhibited completely the same immunoreactivities for these five molecules. Accordingly, the present results give credence to a consideration that the gustducin-immunnoreactive cells in both locations are identical in function(s) e.g., chemo-reception.     

Chronic hypoxia alters calbindin D-28k immunoreactivity in lingual and laryngeal taste buds in the rat

The distribution and abundance of the calcium binding protein, calbindin D-28k (CB) immunoreactivity in the taste buds of the circumvallate papillae and larynx were compared between normoxic and chronically hypoxic rats (10% O2 for 8 weeks). In the normoxic rats, CB immunoreactivity was observed in some cells and fibers of the intragemmal region of the taste buds in the circumvallate papillae. In contrast, in the subgemmal region of the laryngeal taste buds, fibers but not cells were immunoreactive for CB. In chronically hypoxic rats, CB immunoreactive cells and fibers in the taste buds were decreased in the circumvallate papillae. In the laryngeal taste buds, the density of the subgemmal CB immunoreactive fibers in chronically hypoxic rats was greater than in normoxic rats. It is considered that function of the laryngeal taste buds is different from that of the lingual taste buds, so that laryngeal taste buds may be involved in chemosensation other than taste. The altered density of CB immunoreactive cells and fibers in the lingual and laryngeal taste buds is a predominant feature of hypoxic adaptation, and chronic hypoxic exposure might change the chemical sensitivity of the circumvallate papillae and larynx through the regulation of intracellular Ca2+.     

Immunohistochemical characterization of brush cells in the rat larynx

The immunohistochemical characteristics of brush cells in the laryngeal mucosa were examined using immunohistochemistry for various immunohistochemical cell markers including villin at the light and electron microscopic levels. Cells that were immunoreactive to villin were barrel-shaped with thick cytoplasmic processes extending toward the lumen of the laryngeal cavity. Immunoelectron microscopic observations revealed thick and short microvilli with long rootlets of microfilaments. Numerous small clear vesicles and small finger-like cytoplasmic processes were observed in the apical process and lateral membrane, respectively. Double immunofluorescence showed villin-immunoreactive cells were not immunoreactive for the markers of solitary chemosensory cells, GNAT3 and phospholipase C, β2-subunit (PLCβ2), or for that of neuroendocrine cells, synaptosome-associated protein 25kD. Furthermore, immunoreactivities for cytokeratin 18 (CK18) and doublecortin like-kinase 1 in the perinuclear cytoplasm of villin-immunoreactive cells. However, some CK18-immunoreactive cells were immunoreactive to GNAT3 but not to villin. Regarding sensory innervation, only a few intraepithelial nerve endings with P2X3, SP, or CGRP immunoreactivity attached to villin-immunoreactive cells. In the present study, brush cells in the rat laryngeal mucosa were classified by immunoreactivity for villin, and were independent of other non-ciliated epithelial cells such as solitary chemosensory cells and neuroendocrine cells.     

Rat gustatory neurons in the geniculate ganglion express glutamate receptor subunits

Taste receptor cells are innervated by primary gustatory neurons that relay sensory information to the central nervous system. The transmitter(s) at synapses between taste receptor cells and primary afferent fibers is (are) not yet known. By analogy with other sensory organs, glutamate might a transmitter in taste buds. We examined the presence of AMPA and NMDA receptor subunits in rat gustatory primary neurons in the ganglion that innervates the anterior tongue (geniculate ganglion). AMPA and NMDA type subunits were immunohistochemically detected with antibodies against GluR1, GluR2, GluR2/3, GluR4 and NR1 subunits. Gustatory neurons were specifically identified by retrograde tracing with fluorogold from injections made into the anterior portion of the tongue. Most gustatory neurons in the geniculate ganglion were strongly immunoreactive for GluR2/3 (68%), GluR4 (78%) or NR1 (71%). GluR1 was seen in few cells (16%). We further examined if glutamate receptors were present in the peripheral terminals of primary gustatory neurons in taste buds. Many axonal varicosities in fungiform and vallate taste buds were immunoreactive for GluR2/3 but not for NR1. We conclude that gustatory neurons express glutamate receptors and that glutamate receptors of the AMPA type are likely targeted to synapses within taste buds.     

Calbindin D-28k immunoreactive nerve fibers in the carotid body of normoxic and chronically hypoxic rats

The distribution and ultrastructural characteristics of calbindin D-28k immunoreactive nerve fibers were examined in the carotid body of the normoxic control rats by light and electron microscopy, and the abundance of calbindin D-28k fibers in the carotid body was compared in normoxic and chronically hypoxic rats (10% O2 and 3.0-4.0% CO2 for 3 months). Calbindin D-28k immunoreactivity was recognized in nerve fibers within the carotid body. Calbindin D-28k immunoreactive nerve fibers appeared as thin processes with many varicosities. They were distributed around clusters of glomus cells, and around blood vessels. Immunoelectron microscopy revealed that the calbindin D-28k immunoreactive nerve terminals are in close apposition with the glomus cells, and membrane specialization is visible in some terminals. Some dense-cored vesicles in the glomus cells were aggregated in this contact region. The chronically hypoxic carotid bodies were found to be enlarged several fold, and a relative abundance of calbindin D-28k fibers was lesser than in the normoxic carotid bodies. When expressed by the density of varicosities per unit area of the parenchyma, the density of calbindin D-28k fibers associated with the glomus cells in chronically hypoxic carotid bodies was decreased by 70%. These immunohistochemical findings indicate a morphological basis for involvement of calcium binding protein in the neural pathway that modulates carotid body chemoreception.     

Identification of the populations of enteric neurons that have NK1 tachykinin receptors in the guinea-pig small intestine

Simultaneous immunofluorescence labelling was used to investigate the patterns of colocalisation of the NK1 tachykinin receptor with other neuronal markers, and hence determine the functional classes of neuron that bear the NK1 receptor in the guinea-pig ileum. In the myenteric plexus, 85% of NK1 receptor-immunoreactive (NK1r-IR) nerve cells had nitric oxide synthase (NOS) immunoreactivity and the remaining 15% were immunoreactive for choline acetyltransferase (ChAT). Of the latter group, about 50% were immunoreactive for both neuropeptide Y (NPY) and somatostatin (SOM), and had the morphologies of secretomotor neurons. Many of the remaining ChAT neurons were immunoreactive for calbindin or tachykinins (TK), but not both. These calbindin immunoreactive neurons had Dogiel type II morphology. No NK1r-IR nerve cells in the myenteric plexus had serotonin or calretinin immunoreactivity. In the submucosal ganglia, 84% of NK1r-IR nerve cells had neuropeptide Y immunoreactivity and 16% were immunoreactive for TK. It is concluded that NK1r-IR occurs in five classes of neuron; namely, in the majority of NOS-immunoreactive inhibitory motor neurons, in ChAT/TK-immunoreactive excitatory neurons to the circular muscle, in all ChAT/NPY/SOM-immunoreactive secretomotor neurons, in a small proportion of ChAT/calbindin myenteric neurons, and in about 50% of ChAT/TK submucosal neurons.     

Choline acetyltransferase immunoreactivity of putative intrinsic primary afferent neurons in the rat ileum

The colocalisation of choline acetyltransferase (ChAT) with markers of putative intrinsic primary afferent neurons was determined in whole-mount preparations of the myenteric and submucosal plexuses of the rat ileum. In the myenteric plexus, prepared for the simultaneous localisation of ChAT and nitric oxide synthase (NOS), all nerve cells were immunoreactive (IR) for ChAT or NOS, but seldom for both; only 1.6 +/- 1.8% of ChAT-IR neurons displayed NOS-IR and, conversely, 2.8 +/- 3.3% of NOS-IR neurons were ChAT-IR. In preparations double labelled for NOS-IR and the general nerve cell marker, neuron-specific enolase, 24% of all nerve cells were immunoreactive for NOS, indicating that about 75% of all nerve cells have ChAT-IR. All putative intrinsic primary afferent neurons in the myenteric plexus, identified by immunoreactivity for the neurokinin 1 (NK1) receptor and the neurokinin 3 (NK3) receptor, were ChAT-IR. Conversely, of the ChAT-IR nerve cells, about 45% were putative intrinsic primary afferent neurons (this represents 34% of all nerve cells). The cell bodies of putative intrinsic primary afferent neurons had Dogiel type II morphology and were also immunoreactive for calbindin. All, or nearly all, nerve cells in the submucosal plexus were immunoreactive for ChAT. About 46% of all submucosal nerve cells were immunoreactive for both neuropeptide Y (NPY) and calbindin; 91.8 +/- 10.5% of NPY/calbindin cells were also ChAT-IR and 99.1 +/- 0.7% were NK3 receptor-IR. Of the nerve cells with immunoreactivity for ChAT, 44.3 +/- 3.8% were NPY-IR, indicating that about 55% of submucosal nerve cells had ChAT but not NPY-IR. Only small proportions of the ChAT-IR, non-NPY, nerve cells had NK3 receptor or calbindin-IR. It is concluded that about 45% of submucosal nerve cells are ChAT/calbindin/NPY/VIP/NK3 receptor-IR and are likely to be secretomotor neurons. Most of the remaining submucosal nerve cells are immunoreactive for ChAT, but their functions were not deduced. They may include the cell bodies of intrinsic primary afferent neurons.     

Evolutionary origins of taste buds: phylogenetic analysis of purinergic neurotransmission in epithelial chemosensors

Taste buds are gustatory endorgans which use an uncommon purinergic signalling system to transmit information to afferent gustatory nerve fibres. In mammals, ATP is a crucial neurotransmitter released by the taste cells to activate the afferent nerve fibres. Taste buds in mammals display a characteristic, highly specific ecto-ATPase (NTPDase2) activity, suggesting a role in inactivation of the neurotransmitter. The purpose of this study was to test whether the presence of markers of purinergic signalling characterize taste buds in anamniote vertebrates and to test whether similar purinergic systems are employed by other exteroceptive chemosensory systems. The species examined include several teleosts, elasmobranchs, lampreys and hagfish, the last of which lacks vertebrate-type taste buds. For comparison, Schreiner organs of hagfish and solitary chemosensory cells (SCCs) of teleosts, both of which are epidermal chemosensory end organs, were also examined because they might be evolutionarily related to taste buds. Ecto-ATPase activity was evident in elongate cells in all fish taste buds, including teleosts, elasmobranchs and lampreys. Neither SCCs nor Schreiner organs show specific ecto-ATPase activity, suggesting that purinergic signalling is not crucial in those systems as it is for taste buds. These findings suggest that the taste system did not originate from SCCs but arose independently in early vertebrates.     

Dimerization and actin-bundling properties of villin and its role in the assembly of epithelial cell brush borders

Villin is a major actin-bundling protein in the brush border of epithelial cells. In this study we demonstrate for the first time that villin can bundle actin filaments using a single F-actin binding site, because it has the ability to self-associate. Using fluorescence resonance energy transfer, we demonstrate villin self-association in living cells in microvilli and in growth factor-stimulated cells in membrane ruffles and lamellipodia. Using sucrose density gradient, size-exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight, the majority of villin was identified as a monomer or dimer. Villin dimers were also identified in Caco-2 cells, which endogenously express villin and Madin-Darby canine kidney cells that ectopically express villin. Using truncation mutants of villin, site-directed mutagenesis, and fluorescence resonance energy transfer, an amino-terminal dimerization site was identified that regulated villin self-association in parallel conformation as well as actin bundling by villin. This detailed analysis describes for the first time microvillus assembly by villin, redefines the actin-bundling function of villin, and provides a molecular mechanism for actin bundling by villin, which could have wider implications for other actin cross-linking proteins that share a villin-like headpiece domain. Our study also provides a molecular basis to separate the morphologically distinct actin-severing and actin-bundling properties of villin.     

Calbindin immunoreactivity of enteric neurons in the guinea-pig ileum

Previous studies have identified Dogiel type II neurons with cell bodies in the myenteric plexus of guinea-pig ileum to be intrinsic primary afferent neurons. These neurons also have distinctive electrophysiological characteristics (they are AH neurons) and 82-84% are immunoreactive for calbindin. They are the only calbindin-immunoreactive neurons in the plexus. Neurons with analogous shape and electrophysiology are found in submucosal ganglia, but, with antibodies used in previous studies, they lack calbindin immunoreactivity. An antiserum that is more effective in revealing calbindin in the guinea-pig enteric nervous system has been reported recently. In the present work, we found that this antiserum reveals the same population that was previously identified in myenteric ganglia, and does not reveal any further population of myenteric nerve cells. In submucosal ganglia, 9-10% of nerve cells were calbindin immunoreactive with this antiserum. The submucosal neurons with calbindin immunoreactivity were also immunoreactive for choline acetyltransferase, but not for neuropeptide Y (NPY) or vasoactive intestinal peptide (VIP). Small calbindin-immunoreactive neurons (average profile 130 microm2) were calretinin immunoreactive, whereas the large calbindin-immunoreactive neurons (average profile 330 microm2) had tachykinin (substance P) immunoreactivity. Calbindin immunoreactivity was seen in about 50% of the calretinin neurons and 40% of the tachykinin-immunoreactive submucosal neurons. It is concluded that, in the guinea-pig ileum, only one class of myenteric neuron, the AH/Dogiel type II neuron, is calbindin immunoreactive, but, in the submucosal ganglia, calbindin immunoreactivity occurs in cholinergic, calretinin-immunoreactive, secretomotor/vasodilator neurons and AH/Dogiel type II neurons.     

Expression of Calcium Binding Proteins in Mouse Type II Taste Cells

It is well established that calcium is a critical signaling molecule in the transduction of taste stimuli within the peripheral taste system. However, little is known about the regulation and termination of these calcium signals in the taste system. The authors used Western blot, immunocytochemical, and RT-PCR analyses to evaluate the expression of multiple calcium binding proteins in mouse circumvallate taste papillae, including parvalbumin, calbindin D28k, calretinin, neurocalcin, NCS-1 (or frequenin), and CaBP. They found that all of the calcium binding proteins they tested were expressed in mouse circumvallate taste cells with the exception of NCS-1. The authors correlated the expression patterns of these calcium binding proteins with a marker for type II cells and found that neurocalcin was expressed in 80% of type II cells, whereas parvalbumin was found in less than 10% of the type II cells. Calretinin, calbindin, and CaBP were expressed in about half of the type II cells. These data reveal that multiple calcium binding proteins are highly expressed in taste cells and have distinct expression patterns that likely reflect their different roles within taste receptor cells.     

Cerebellar localization and colocalization of GABA and calcium binding protein-D28K

Immunocytochemical studies using antibodies raised against the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) and against the 28 Kd vitamin D dependent calcium binding protein (calbindin) in the cerebellum, are reviewed. The GABA immunoreactive neurones found in the cerebellar cortex were the Purkinje cell (PC), the three classes of intrinsic inhibitory interneurones, stellate, basket and Golgi cells and the cells of Lugaro. Some of the neurons of the cerebellar nuclei were also found to be GABA immunoreactive. A part of these could be identified as extrinsic neurones projecting either back to the cerebellar cortex, or to the inferior olive, both these pathways being topographically highly organized but arising from independent parent neurons. The presumed inhibitory function of these two pathways are discussed. Calbindin immunoreactivity in the cerebellum was confined to the PCs, staining concerned the whole cell including soma, branching dendrites, axons and axons terminals. The antibody, which appears to be tightly bound to the PC in vivo, failed to stain some of the PC when cerebellar slices maintained in vitro were studied. The stability of the antigen-antibody binding and the use of calbindin as a marker specific for the PC in the cerebellum, is discussed. Co-localization of GABA with calbindin as well as with other calcium binding proteins are reported to be found in the PCs. While these co-localizations have led to much speculation, conclusive functional roles for them have not been identified at present.     

Localisation of actin, villin, fimbrin, ezrin and ankyrin in rat taste receptor cells

Mammalian taste buds consist of 50–150 pear- or spindle-shaped taste receptor cells which contain, at their apical cell surface, a bundle of microvillar projections. The microvilli probably serve to increase the receptive membrane surface of the chemosensory receptor cells. The molecular basis controlling the ultrastructure of taste receptor microvilli is present unknown. In the present study we analysed, by immunostaining at the light and electron microscopic levels and by immunoblotting, components of the cytoskeleton of these microvilli. We show here that taste cell microvilli contain the major cytoskeletal proteins of intestinal microvilli, actin, fimbrin and villin. Another actin-binding, peripheral membrane protein of intestinal microvilli, ezrin, was also localised to taste cell microvilli, where ezrin might play a role, for example, in placement of specific membrane proteins to the microvillus membrane. In search of further linkage proteins, we found ankyrin localised along the basolateral cell surface of taste receptor cells, where ankyrin might be involved in the immobilisation of the Na⁺, K⁺-ATPase or other ion-translocating proteins of taste cells to the membrane cytoskeleton. Accepted: 26 April 1999     

Keratin 19-like immunoreactivity in receptor cells of mammalian taste buds

Three monoclonal antibodies, 4.62, LPZK and 170.2.14, were usedto evaluate keratin 19-like immunoreactivity in gustatory epithelia.Keratin 19-like immunoreactivity was restricted to the intragemmalcells for all types of mammalian taste buds examined. Thesetaste buds included fungiform, foliate and vallate taste budsin rat, gerbil and rabbit, and nasopalatine, epiglottal andpalatine taste buds in rat. There was no keratin 19-like immunoreactivityin basal cells or in perigemmal cells lateral to the immunoreactivetaste receptor cells. Denervation of the rat vallate papillaeliminated all taste buds, as well as all immunoreactive tastecells. That the immunoreactive material in the taste cells waskeratin 19 was supported by the comparable staining of rat tastebuds with each of three monoclonal antibodies specific for keratin19. Furthermore, as predicted, these antibodies selectivelystained luminal cells of ral bile ducts, bladder, salivary ducts,trachea, ureter and uterus. It was concluded that monoclonalantibodies against keratin 19 can usefully distinguish intragemmaltaste receptor cells from keratinocytes, and from the perigemmaland basal cells of gustatory epithelia. Anti-keratin 19 antibodiesmay serve to identify differentiated taste cells in gustatoryepithelia undergoing taste bud development, renewal, degenerationor regeneration.     

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.     

Villin-like actin-binding proteins are expressed ubiquitously in Arabidopsis

In an attempt to elucidate the biological function of villin-like actin-binding proteins in plants we have cloned several genes encoding Arabidopsis proteins with high homology to animal villin. We found that Arabidopsis contains at least four villin-like genes (AtVLNs) encoding four different VLN isoforms. Two AtVLN isoforms are more closely related to mammalian villin in their primary structure and are also antigenically related, whereas the other two contain significant changes in the C-terminal headpiece domain. RNA and promoter/beta-glucuronidase expression studies demonstrated that AtVLN genes are expressed in all organs, with elevated expression levels in certain types of cells. These results suggest that AtVLNs have less-specialized functions than mammalian villin, which is found only in the microvilli of brush border cells. Immunoblot experiments using a monoclonal antibody against pig villin showed that AtVLNs are widely distributed in a variety of plant tissues. Green fluorescent protein fused to full-length AtVLN and individual AtVLN headpiece domains can bind to both animal and plant actin filaments in vivo.     

Localization of 28 kDa calbindin in human odontoblasts

Summary The presence of 28 kDa calbindin in human odontoblasts was studied by use of specific antibodies raised against chick duodenal 28 kDa calbindin, in immunofluorescence, immuno-peroxidase, and electron-microscopic labelling experiments.The calbindin-like protein was detected mainly in the cytoplasm of odontoblast cell bodies, in their processes and occasionally in their nuclei. Correspondingly, at the ultrastructural level, immunoreactive material was associated with the cytosol, microfilaments and cilia. These findings suggest that human odontoblasts express a 28 kDa vitamin D-dependent calcium-binding protein, unlike those of rats and mice in which ameloblasts are the only cells immunoreactive for the protein.