Regulation of α-Transducin and α-Gustducin Expression by a High Protein Diet in the Pig Gastrointestinal Tract.

BACKGROUND: The expression of taste receptors (TASRs) and their signalling molecules in the gastrointestinal (GI) epithelial cells, including enteroendocrine cells (EECs), suggests they participate in chemosensing mechanisms influencing GI physiology via the release of endocrine messengers. TASRs mediate gustatory signalling by interacting with different transducers, including α-gustducin (Gαgust) and α-transducin (Gαtran) G protein subunits. This study tested whether Gαtran and Gαgust immunoreactive (-IR) cells are affected by a short-term (3 days) and long-term (30 days) high protein (Hp) diet in the pig GI tract. RESULT: In the stomach, Gαgust and Gαtran-IR cells contained serotonin (5-HT) and ghrelin (GHR), while in the small and large intestine, Gαgust and Gαtran-IR colocalized with 5-HT-, cholecystokinin (CCK)- and peptide YY (PYY)-IR. There was a significant increase in the density of Gαtran-IR cells in the pyloric mucosa in both short- and long-term Hp diet groups (Hp3 and Hp30) vs. the control group (Ctr) (P<0.05), while the increase of Gαgust-IR cells in the pyloric mucosa was significant in Hp30 group vs. Ctr and vs. Hp3 (P<0.05); these cells included Gαtran / 5HT-IR and Gαtran / GHR-IR cells (P<0.05 and P<0.001 vs. Ctr, respectively) as well as Gαgust /5-HT-IR or Gαgust / GHR-IR cells (P<0.05 and P<0.01 vs. Ctr, respectively). In the small intestine, we recorded a significant increase in Gαtran-IR cells in the duodenal crypts and a significant increase of Gαgust-IR cells in the jejunal crypts in Hp3 group compared to HP30 (P<0.05). With regard to the number of Gαtran-Gαgust IR cells colocalized with CCK or 5-HT, there was only a significant increase of Gαtran / CCK-IR cells in Hp3 group compared to Ctr (P = 0.01). CONCLUSION: This study showed an upregulation of selected subpopulations of Gαgust / Gαtran-IR cells in distinct regions of the pig GI tract by short- and long-term Hp diet lending support to TASR-mediated effects in metabolic homeostasis and satiety mechanisms.

Expression of umami-taste-related genes in the tongue: a pilot study for genetic taste diagnosis.

OBJECTIVE: Expression of taste-related genes in the tongue was analysed to develop a technique for genetic diagnosis of umami taste disorders. MATERIALS AND METHODS: Tissue samples were collected from healthy volunteers by scraping the foliate papillae of the tongue. Immunocytochemistry staining of gustducin, a taste-cell-specific G protein, and gene expression analysis by real-time polymerase chain reaction of ß-actin, gustducin (GNAT3) and umami receptors (T1R1, T1R3 and mGluR1) were performed. Changes in umami receptor expression following application of umami substances onto the tongue were analysed. RESULTS: Gustducin-positive cells were observed in the samples, indicating the presence of taste cells. Gene expression of ß-actin, GNAT3, T1R1 and T1R3 was detected in all seven samples tested, while that of mGluR1 was detected in four samples. Sequence analysis by NCBI Blast showed that each polymerase chain reaction product had a 99% rate of identification of its target sequence. Stimulation of the tongue with monosodium glutamate significantly upregulated the gene expression levels of T1R1 and T1R3, indicating that this method can detect alterations in umami-related gene expression. CONCLUSION: Evaluation of the expression of the umami receptor genes, T1R1 and T1R3, in the tongue may be clinically useful for objective genetic diagnosis of umami taste disorders.

Cryptochrome in sponges: a key molecule linking photoreception with phototransduction.

Sponges (phylum: Porifera) react to external light or mechanical signals with contractile or metabolic reactions and are devoid of any nervous or muscular system. Furthermore, elements of a photoreception/phototransduction system exist in those animals. Recently, a cryptochrome-based photoreceptor system has been discovered in the demosponge. The assumption that in sponges the siliceous skeleton acts as a substitution for the lack of a nervous system and allows light signals to be transmitted through its glass fiber network is supported by the findings that the first spicules are efficient light waveguides and the second sponges have the enzymatic machinery for the generation of light. Now, we have identified/cloned in Suberites domuncula two additional potential molecules of the sponge cryptochrome photoreception system, the guanine nucleotide-binding protein ß subunit, related to ß-transducin, and the nitric oxide synthase (NOS)-interacting protein. Cryptochrome and NOSIP are light-inducible genes. The studies show that the NOS inhibitor L-NMMA impairs both morphogenesis and motility of the cells. Finally, we report that the function of primmorphs to produce reactive nitrogen species can be abolished by a NOS inhibitor. We propose that the sponge cryptochrome-based photoreception system, through which photon signals are converted into radicals, is coupled to the NOS apparatus.

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 Cß2 (PLCß2), 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.

Gustducin is expressed in the taste buds of the chicken.

We investigated the expression of gustducin in chicken taste buds using molecular biological, biochemical and immunohistochemical techniques. Expression of a gustducin-like sequence was detected by RT-PCR in the tissues containing taste buds, and corresponded to the predicted gustducin gene in the chicken. Expression of this sequence was not detected in the brain, heart, liver, pancreas, intestine, kidney and testis of the chicken. The expressed sequence had a high specificity for oral tissues that contained taste buds. These results suggest that the detected sequence was the chicken gustducin gene. Next, we generated a polyclonal antiserum against the chicken gustducin protein to observe its localization in the oral tissues. The results revealed that the chicken gustducin was specifically expressed in the taste buds. It is suggested that the chicken has a gustatory system mediated by gustducin, and chicken gustducin is a reliable marker for taste buds or taste cells. This is the first molecular biological, biochemical and immunohistochemical demonstration of the presence of gustducin in the chicken.

Synaptophysin as a probable component of neurotransmission occurring in taste receptor cells.

Taste signal is received in taste buds and transmitted via sensory afferent nerves to the brainstem. Although a signaling pathway involving phospholipase C-beta2 has been shown to transduce taste signals of bitterness, sweetness and umami in taste receptor cells (Type II cells), these taste receptor cells appear to be different from the presynaptic cells (Type III cells) containing afferent synapses associated with nerve processes. To elucidate the neurotransmission system in the taste receptor cells expressing phospholipase C-beta2, we searched for candidate molecules involved in the neurotransmission, and identified synaptophysin. Synaptophysin was expressed in the taste receptor cells expressing phospholipase C-beta2, as well as in the presynaptic cells harboring synaptic structures with taste nerves and containing serotonin. Synaptophysin-immunoreactive signals were not limited to gustducin-positive bitter taste receptor cells, and sweet/umami taste receptor cells were indicated to also express synaptophysin. Expression of synaptophysin was already initiated 6 days after cell division, almost in synchrony with the initiation of phospholipase C-beta2 expression. Synaptophysin-containing cells co-expressed vesicular-associated membrane protein 2, a v-SNARE molecule which is important for exocytosis. In addition, majority of the synaptophysin-expressing cells also expressed cholecystokinin, a neuropeptide expressed in taste buds. These results suggest that the taste receptor cells have a neurotransmission system involving synaptophysin, which occurs alternatively or additionally to a recently shown hemichannel system.

Interaction of retinal guanylate cyclase with the alpha subunit of transducin: potential role in transducin localization.

Vertebrate phototransduction is mediated by cGMP, which is generated by retGC (retinal guanylate cyclase) and degraded by cGMP phosphodiesterase. Light stimulates cGMP hydrolysis via the G-protein transducin, which directly binds to and activates phosphodiesterase. Bright light also causes relocalization of transducin from the OS (outer segments) of the rod cells to the inner compartments. In the present study, we show experimental evidence for a previously unknown interaction between G(alphat) (the transducin alpha subunit) and retGC. G(alphat) co-immunoprecipitates with retGC from the retina or from co-transfected COS-7 cells. The retGC-G(alphat) complex is also present in cones. The interaction also occurs in mice lacking RGS9 (regulator of G-protein signalling 9), a protein previously shown to associate with both G(alphat) and retGC. The G(alphat)-retGC interaction is mediated primarily by the kinase homology domain of retGC, which binds GDP-bound G(alphat) stronger than the GTP[S] (GTPgammaS; guanosine 5'-[gamma-thio]triphosphate) form. Neither G(alphat) nor G(betagamma) affect retGC-mediated cGMP synthesis, regardless of the presence of GCAP (guanylate cyclase activating protein) and Ca2+. The rate of light-dependent transducin redistribution from the OS to the inner segments is markedly accelerated in the retGC-1-knockout mice, while the migration of transducin to the OS after the onset of darkness is delayed. Supplementation of permeabilized photoreceptors with cGMP does not affect transducin translocation. Taken together, these results suggest that the protein-protein interaction between G(alphat) and retGC represents a novel mechanism regulating light-dependent translocation of transducin in rod photoreceptors.

Native fractionation: isolation of native membrane-bound protein complexes from porcine rod outer segments using isopycnic density gradient centrifugation.

Networks of interacting protein control physiological processes in all living cells. Considerable effort has recently been invested in understanding protein interactions under normal and diseased conditions. One approach to elucidate the composition of protein complexes is native fractionation followed by immunological or MS-based identification of individual compounds. Native fractionation, in contrast to widespread affinity-based purification methods, allows analysis of protein interactions at the endogenous expression level and within a physiological context. In this chapter we describe a protocol for native fractionation of membrane-bound protein complexes from isolated porcine rod outer segments (ROSs). Protein complexes from isolated ROS membranes were solubilized using the nonionic detergent beta-dodecylmaltoside and fractionated by isopycnic sucrose density gradient centrifugation. Immunolabeling of individual sucrose gradient fractions demonstrated colocalization of proteins involved in the phototransduction pathway in photoreceptor outer segments.

Differentiation of alpha-gustducin in taste buds of the mouse soft palate and fungiform papillae.

We used alpha-gustducin, a type II taste-cell-specific G protein, to investigate the onset of taste transduction and its relation to the development of the soft palate (SP) and fungiform (FF) papillae taste buds in the mouse. Paraffin wax embedded sections were prepared from the SP and anterior region of the tongue of the mouse from birth until postnatal day (PD) 63. No alpha-gustducin-immunoreactive cells were observed on the day of birth. One day later, alpha-gustducin was immunolocalised in taste buds with pores with a relatively higher frequency recorded in the SP as compared with the FF papillae. The immunoreactive cells were spindle shaped with elongated processes extending from the base to the pore of the taste buds. On PD 7, the number of taste buds containing alpha-gustducin-immunoreactive cells in the SP was three times greater than that of FF papillae. Our results indicate that taste transduction is essentially acquired from the time of birth. Moreover, the onset of taste transduction by the SP taste buds developed earlier than that achieved by taste buds in the FF papillae.

Subunit dissociation and diffusion determine the subcellular localization of rod and cone transducins.

Activation of rod photoreceptors by light induces a massive redistribution of the heterotrimeric G-protein transducin. In darkness, transducin is sequestered within the membrane-enriched outer segments of the rod cell. In light, it disperses throughout the entire neuron. We show here that redistribution of rod transducin by light requires activation, but it does not require ATP. This observation rules out participation of molecular motors in the redistribution process. In contrast to the light-stimulated redistribution of rod transducin in rods, cone transducin in cones does not redistribute during activation. Remarkably, when cone transducin is expressed in rods, it does undergo light-stimulated redistribution. We show here that the difference in subcellular localization of activated rod and cone G-proteins correlates with their affinity for membranes. Activated rod transducin releases from membranes, whereas activated cone transducin remains bound to membranes. A synthetic peptide that dissociates G-protein complexes independently of activation facilitates dispersion of both rod and cone transducins within the cells. This peptide also facilitates detachment of both G-proteins from the membranes. Together, these results show that it is the dissociation state of transducin that determines its localization in photoreceptors. When rod transducin is stimulated, its subunits dissociate, leave outer segment membranes, and equilibrate throughout the cell. Cone transducin subunits do not dissociate during activation and remain sequestered within the outer segment. These findings indicate that the subunits of some heterotrimeric G-proteins remain associated during activation in their native environments.

Secretory cells of the airway express molecules of the chemoreceptive cascade.

Airway secretion is maintained by specialized non-ciliated epithelial cells whose phenotype varies with their topographical location. In addition, specialized epithelial cells located in the airway contain the molecular machinery of chemoreceptive elements. Our aim has been to evaluate whether the secretory cells themselves possess a chemoreceptive capability, which requires the simultaneous presence of chemosensory and secretory mechanisms. We performed immunohistochemical analysis with antibodies against the Clara-cell-specific secretory proteins, CC10 and CC26, as secretory markers. As chemoreceptive markers, we employed antibodies against alpha-gustducin and phospholipase C beta 2 (PLCbeta2), two components of the taste transduction pathway. We also attempted to characterize further the secretory cell type by using a marker of chloride secretion, cystic fibrosis transmembrane regulator (CFTR). We found alpha-gustducin localized in non-ciliated cells of the epithelium lining the trachea and bronchioles of adult rats, where it was also co-expressed with CC10 and CC26. Ultrastructural immunohistochemistry revealed alpha-gustducin in the apical cytoplasm of secretory cells, concentrated around and inside the granules. CFTR was also observed in a subpopulation of non-ciliated epithelial cells, co-localized with some alpha-gustducin- and PLCbeta2-immunoreactive cells, at all levels of the airway epithelium. We conclude that non-ciliated epithelial cells of the rat airway express components of distinct signaling mechanisms and suggest that secretory events are driven by a molecular mechanism activated by the binding of luminal substances to G-protein-coupled receptors.

Structure of bovine fungiform taste buds and their immunoreactivity for gustducin.

The taste buds of bovine fungiform papillae were studied by light and electron microscopy using both histological and immunohistochemical methods. The taste buds existed in the epithelium of the apical region of the papillae. By electron microscopy, two types of taste cells, namely type I and type II cells, could be classified according to the presence of dense-cored vesicles, the cytoplasmic density and the cell shape. Type I cells were thin, had an electron-dense cytoplasm containing dense-cored vesicles, and possessed long thick apical processes in the taste pore. Type II cells were thick, had an electron-lucent cytoplasm containing many electron-lucent vesicles, rather than dense-cored vesicles, and possessed microvilli in the taste pore. Immunohistochemical staining with an antiserum against gustducin was investigated by both light and electron microscopy using the avidin-biotin complex (ABC) method. Some, but not all, of the type II cells exhibited gustducin immunoreactivity, whereas none of the type I cells showed any immunoreactivity.

Transducin subunit stoichiometry and cellular distribution in rod outer segments.

Transducin is a heterotrimeric GTP-binding protein found in the outer segment of vertebrate retinas that links the photoactivation of rhodopsin (R*) with activation of a robust type VI cGMP phosphodiesterase (PDE6). Association of the alpha subunit of Transducin (G(alphat)) with the beta-gamma complex (G(betagamma)) is necessary for interaction of the holoprotein with R* and exchange of a GTP for a previously bound GDP. We have investigated the abundances of the three Transducin subunits by eluting them from bovine rod outer segment membranes by centrifugation under various conditions in vitro. We find that a substantial amount of G(betagamma) is eluted from ROS under conditions that do not elute G(alphat) and that there is an overall three to fourfold molar excess of G(betagamma) to G(alphat) in rod outer segments. These results suggest that the production and/or turnover of G(alphat), G(beta), and G(gamma) in the rod outer segment are controlled independently.

Evidence of solitary chemosensory cells in a large mammal: the diffuse chemosensory system in Bos taurus airways.

The diffuse chemosensory system (DCS) of the respiratory apparatus is composed of solitary chemosensory cells (SCCs) that resemble taste cells but are not organized in end organs. The discovery of the DCS may open up new approaches to respiratory diseases. However, available data on mammalian SCCs have so far been collected from rodents, the airways of which display some differences from those of large mammals. Here we investigated the presence of the DCS and of SCCs in cows and bulls (Bos taurus), in which the airway cytology is similar to that in humans, focusing our attention on detection in the airways of molecules involved in the transduction cascade of taste [i.e. alpha-gustducin and phospholipase C of the beta2 subtype (PLCbeta2)]. The aim of the research was to extend our understanding of airway chemoreceptors and to compare the organization of the DCS in a large mammal with that in rodents. Using immunocytochemistry for alpha-gustducin, the taste buds of the tongue and arytenoid were visualized. In the trachea and bronchi, alpha-gustducin-immunoreactive SCCs were frequently found. Using immunocytochemistry for PLCbeta2, the staining pattern was generally similar to those seen for alpha-gustducin. Immunoblotting confirmed the expression of alpha-gustducin in the tongue and in all the airway regions tested. The study demonstrated the presence of SCCs in cows and bulls, suggesting that DCSs are present in many mammalian species. The description of areas with a high density of SCCs in bovine bronchi seems to indicate that the view of the DCS as made up of isolated cells totally devoid of ancillary elements is probably an oversimplification.

Solitary chemoreceptor cell proliferation in adult nasal epithelium.

Nasal trigeminal chemosensitivity in mice and rats is mediated in part by solitary chemoreceptor cells (SCCs) in the nasal epithelium (Finger et al., 2003). Many nasal SCCs express the G-protein alpha-gustducin as well as other elements of the bitter-taste signaling cascade including phospholipase Cbeta2, TRPM5 and T2R bitter-taste receptors. While some populations of sensory cells are replaced throughout life (taste and olfaction), others are not (hair cells and carotid body chemoreceptors). These experiments were designed to test whether new SCCs are generated within the epithelium of adult mice. Wild type C57/B6 mice were injected with the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) to label dividing cells. At various times after injection (1-40 days), the mice were perfused with 4% paraformaldehyde and prepared for dual-label immunocytochemistry. Double labeled cells were detected as early as 3 days post BrdU injection and remained for as long as 12 days post-injection suggesting that SCCs do undergo turnover like the surrounding nasal epithelium. No BrdU labeled cells were detected after 24 days suggesting relatively rapid replacement of the SCCs.

Changes in rhodopsin kinase and transducin in the rat retina in early-stage diabetes.

To establish changes in phototransduction in diabetes, the effects of high glucose on rhodopsin kinase (RK) and transducin (G(t)), as well as recoverin, were examined in the retina of STZ-induced diabetic rats. Diabetes was induced by single intraperitoneal injection of STZ (50mg/kg) to Sprague-Dawley (SD) rats and the animals were sacrificed after 6 weeks. Immunohistochemistry (IHC) and Western blot analysis were carried out using antibodies against RK and G(talpha) (alpha subunit of G(t)) in the STZ-induced diabetic retina and the control retina. The expression level of recoverin protein was also analysed. In the diabetic retina, while the expression of RK protein increased, that of G(talpha) and recoverin proteins decreased. RK immunoreactivity (IR) appeared generally in the retina, and its signal increased in the outer limiting membrane (OLM), some rod cells in the outer segment layer (OSL) and at the tip of the outer plexiform layer (OPL) in the diabetic retina. G(talpha)-IR also appeared in the OPL and in photoreceptor layer. In the diabetic retina, G(talpha)-IR significantly decreased in the OPL, indicating RK-IR increase. This study illustrates the alterations in RK, G(talpha) and recoverin in the diabetic retina that may induce dysfunctions in phototransduction even in early-stage diabetes.

[Age-related changes in renewal of taste bud cells and expression of taste cell-specific proteins in mice].

It is known that taste sensitivities change throughout life and they decline with aging. We have also found that both detection and recognition thresholds of taste were significantly elevated in aged persons, while oral somatic sensations hardly changed. To elucidate the peripheral mechanisms of the decline of taste sensitivities with age, we first investigated age-associated changes in cell renewal of taste buds in the circumvallate papillae of ddY mice, because one of the major characteristic differences of taste receptors from somatosensory receptors is a continual turnover of cells. In addition, we examined the expression patterns of taste cell-specific proteins such as protein gene products 9.5 (PGP 9.5) and gustducin, and ultrastructure of taste buds which may change resulting from the change of turnover rate. As a reference, we also used mice in the developing stage. The rate of cell renewal was examined using 5-bromo-2'-deoxyuridine (BrdU), which is incorporated in DNA during the S-phase of cells. BrdU and the proteins were detected immunohistochemically and the ultrastructure was investigated by electron microscopy. The aged mice demonstrated a delayed cell renewal and highly vacuolated cytoplasm in taste buds, while they showed no change in PGP 9.5-immunoreactive cells and an increase in gustducin-immunoreactive cells compared with young adult mice. In contrast, the developing mice showed a higher rate of cell turnover and rapid increase of PGP 9.5- and gustducin-immunoreactive cells reaching the mature level in 3 weeks after birth. The changes observed in the present study in aged mice may be related to the decline in taste sensitivity with aging.

The rod photoreceptor-specific nuclear receptor Nr2e3 represses transcription of multiple cone-specific genes.

This study addresses one genetic regulatory mechanism that establishes the distinct identities of rod and cone photoreceptors. Previous work has shown that mutations in either humans or mice in the gene coding for photoreceptor-specific nuclear receptor Nr2e3 cause a progressive retinal degeneration characterized by increased numbers of short-wave cones. In the present work, we have examined the cellular and developmental pattern of Nr2e3 protein localization in mammals and fish, identified an optimal Nr2e3 DNA-binding site using cycles of binding to recombinant Nr2e3, characterized the transcriptional activity of wild type and one of the disease-associated point mutations in Nr2e3 in transfected cells, and characterized the transcriptional defects in the naturally occurring Nr2e3 mutant (rd7) mouse. These experiments indicate that in the mature vertebrate retina Nr2e3 is expressed exclusively in rods, that expression of Nr2e3 is one of the earliest events in the pathway of rod-specific photoreceptor development, and that Nr2e3 functions, either directly or indirectly, as a repressor of cone-specific genes in rod photoreceptor cells.

Top-down analysis of protein isoprenylation by electrospray ionization hybrid quadrupole time-of-flight tandem mass spectrometry; the mouse Tgamma protein.

Protein isoprenylation, an important post-translational modification with a lipid, involves the selective attachment of two types of isoprenoids, farnesyl (C15) and geranylgeranyl (C20). The isoprenoid is linked via a thioether bond to the C-terminal cysteine residue of a variety of cellular proteins, including the heterotrimeric G protein gamma-subunits. One member of the G protein family, transducin (Talpha/Tbetagamma), plays a central role in visual transduction, and the structure-function relationship has been extensively studied with purified proteins, predominantly with bovine transducin that was shown to be farnesylated at the C-terminal cysteine residue of the gamma-subunit (Tgamma). We report here the structure of the C-terminal modification of mouse Tgamma, which has not yet been elucidated owing to the low amount of protein that can be isolated from the mouse retina. Electrospray ionization mass spectrometry (ESI-MS) of the high-performance liquid chromatography (HPLC)-purified Tgamma was in good agreement with the calculated mass of the farnesylated and methylated form of mouse Tgamma (Pro1-Cys70). A 'top-down' analysis of intact Tgamma using an ESI hybrid quadrupole time-of-flight (TOF) tandem mass spectrometer provided isoprenyl-specific ions that were observed to produce ions separated by 204 Da from the conventional (unmodified) precursor ion or the C-terminal sequence ions. Such characteristic fragmentation on an isoprenoid observed in top-down analysis could be useful in general for determining the type of isoprenylation as well as probing the site of modification in the protein sequence.