The a-subunit of the trimeric G-protein complex specific for taste receptor cells of the tongue, a-gustducin, is described here to be also expressed in the stomach and intestine. The a-gustducin-containing cells were identified as brush cells that are scattered throughout the surface epithelium of the gut and share structural features of taste receptor cells of the tongue. These findings provide clues to the long-sought molecular and cellular basis for chemoreception in the gut.It is generally believed that the epithelium lining the inner surface of the gut can sense chemical components of the lumenal contents. This chemosensory information appears to be important for the regulation of various aspects of gastrointestinal secretion, resorption, and motility (1, 2). Classical examples of intestinal chemosensitivity are the dependence of gastric emptying on the chemical nature of the nutrients present in the small intestine and the involvement of chemical preabsorption information in short-term regulation of food intake (2). The cellular and molecular basis for chemoreception in the gut is hitherto unknown. In this study we addressed the question ofwhether the epithelium of the gut might express a-gustducin, the GTP-binding a-subunit of a trimeric Gprotein complex that is specific for taste receptor cells of the tongue (3). Here we show that a-gustducin is also expressed in the epithelium of the gut where it is associated with a specialized cell type long known under the names brush cell, tufted cell, or caveolated cell (4-6). The function of this cell type, which is present in humans, rats, and probably all other mammals, had been enigmatic until now. MATERIALS AND METHODSAntibodies and Immunostaining. A polyclonal antibody specific for a-gustducin was raised in a rabbit immunized with a synthetic peptide comprising amino acid residues 92-113 of the rat a-gustducin sequence (3). This sequence stretch is unique for a-gustducin and is not present in the sequences of any other known G-protein. Antibodies were affinity-purified to the peptide adsorbed to nitrocellulose (7,8). Polyclonal rabbit antibodies specific for chromogranin A and serotonin (9) and mouse monoclonal antibodies to villin (Dianova, Hamburg, Germany) and cytokeratin 18 (Progen, Heidelberg) were also used in this study. Indirect immunofluorescence was applied to 1-,um thick tissue sections of quick-frozen and Epon-embedded tissues as described (8). For doubleimmunofluorescence sections were incubated with a mixture of the rabbit antibody against a-gustducin and mouse monoclonal antibodies either specific for villin or cytokeratin 18. Primary antibodies were diluted with PBS: anti-gustducin (1:200), anti-chromogranin (1:4,000), anti-serotonin (1:10,000), anti-villin (0.1 /Lg/ml-1), anti-cytokeratin 18 (0.5 ,ug/ml-1). As secondary antibodies fluorescein isothiocyanate-labeled goat anti-mouse IgG and tetramethylrhodamine isothiocyanate-labeled goat anti-rabbit IgG (Dianova) were used at concentrations of 0.1 Ag/ml-1.Immunoblotting. Various tissue...

The major pancreatic excretory ducts have been shown to contain a large number of specialized epithelial cells, named brush cells, that are characterized by an apical tuft of stiff microvilli. The function of pancreatic brush cells is unknown. Because of some structural similarities to taste receptor cells of the tongue, we addressed the question whether pancreatic brush cells contain the taste cell-specific GTP-binding protein, alpha-gustducin, and hence might be considered to be involved in intraductal chemoreception. By immunostaining, we show that ductal brush cells of the rat pancreatic duct system contain alpha-gustducin, which is concentrated in the apical tuft of microvilli and is also found along the basolateral cell surface. A further outcome of this study is that brush cells are concentrated in the terminal portions of extralobular ducts and in the major pancreatic duct where brush cells comprise up to 22% of the ductal epithelium. Immunoblotting of the major pancreatic duct revealed a 42-kDa band that comigrated with alpha-gustducin of the rat tongue. In view of our previous observation that the ductal brush cells are particularly rich in nitric oxide synthase-I, there is reason to assume that these cells might play a role in certain aspects of chemoreceptive signalling. Thus, chemosensory control of pancreatic secretion might occur at two independent sites, the intestine and the terminal portions of the excretory duct system.

The ability of the gut mucosa to sense the chemical composition of chyme is important for gastrointestinal functions. The demonstration of gustducin and transducin, two alpha-subunits of GTP-binding proteins involved in gustatory signal transduction, in gastrointestinal epithelial cells provides first clues to the molecular basis of enteric chemosensitivity. Nitric oxide may play a role as a secondary messenger.

Abstract. The apex of hair cells of the chicken auditory organ contains three different kinds of assemblies of actin filaments in close spatial proximity. These are (a) paracrystals of actin filaments with identical polarity in stereocilia, (b) a dense gellike meshwork of actin filaments forming the cutieular plate, and (c) a bundle of parallel actin filaments with mixed polarities that constitute the circumferential filament belt attached to the cytoplasmic aspect of the zonula adhaerens (ZA). Each different supramolecular assembly of actin filaments contains a specific actin filament crosslinking protein which is unique to that particular assembly. Thus fimbrin appears to be responsible for paracrystallin packing of actin filaments in stereocilia;an isoform of speetrin resides in the cuticular plate where it forms the whisker-like crossbridges, and a actinin is the actin crosslinking protein of the circumferential ZA bundle. Tropomyosin, which stabilizes actin filaments, is present in all the actin filament assemblies except for the stereocilia. Another striking finding was that myosin appears to be absent from the ZA ring and cuticular plate of hair cells although present in the ZA ring of supporting cells. The abundance of myosin in the ZA ring of the surrounding supporting cells means that it may be important in forming a supporting tensile cellular framework in which the hair cells are inserted.Oth E of the most fascinating features of cells is that at e same moment in time a cell can carry out a diverse array of activities, most of which involve the cytoskeleton. For example, a cell can maintain static surface projections or microvilli, nearby it can phagocytose particles, and at another location it can move by ameboid motion. Thus, an important problem in cell biology is how the cell controls different assemblies of actin filaments. We have chosen the hair cell of the cochlea as a model system that may help us to understand how different actin assemblies are formed and maintained in the same cell at the same time.Located at the apical end of hair cells in the chick inner ear are three different assemblies of actin filaments. One is a paracrystalline bundle of actin filaments present in each sterexxfilium. These filaments are parallel, have identical polarities, and are maximally crossbridged together (Tilney et al., 1983). The second, the cuticular plate, is a complex actin gel located immediately beneath the stereocilia. Here the actin filaments are randomly oriented relative to each other, yet connected together by tiny whiskerlike strands of proteinaceous material (DeRosier and Tilney, 1989). The third is a circumferential belt of actin filaments encircling the apicolateral margins of the cell at the zonula adhaerens (ZA) 1. The filaments that make up this belt, although paral-1. Abbreviations used in this paper: F-actin, filamentous actin; ZA, zonula adhaerens.lel, are of mixed polarities (Hirokawa and Tflney, 1982). Since these three different assemblies of actin filaments all occur in the same cell...

Brush cells represent a population of epithelial cells with unknown function, which are scattered throughout the epithelial lining of both the respiratory system and the alimentary system. These cells are reliably distinguished from other epithelial cells only at the ultrastructural level by the presence of an apical tuft of stiff microvilli and extremely long microvillar rootlets that may project down to the perinuclear space. In the present study we show that brush cells can be identified in tissue sections even at the light microscopic level by immunostaining with antibodies against villin and fimbrin, two proteins that crosslink actin filaments to form bundles. In brush cells, villin and fimbrin are not only present in the actin filament core bundles of apical microvilli and their long rootlets but, in addition, both proteins are also associated with microvilli extending from the basolateral cell surface of the brush cells. Basolateral immunostaining specific for villin and fimbrin does not occur in any other epithelial cell type of the respiratory and alimentary tract. Thus immunostaining with antibodies against both proteins allows unequivocal identification of individual brush cells even in sectional planes that do not contain the brightly stained apical tuft of microvilli and their long rootlets.