Glucose transporter/T1R3-expressing cells in rat tracheal epithelium.

Glucose transport plays an important role in maintaining low sugar concentration in airway surface liquid (ASL), which is critical for mucociliary clearance and bacterial colonization. Experimental evidence indicates that glucose/hexose uptake in lung/airway cells occurs by means of two structurally distinct glucose transporter pathways: the Na(+) -dependent glucose transporters (SGLT family) and the facilitative glucose transporters (GLUT family). In this study, we examined the expression of the major glucose transporters of the intestine, GLUT2, GLUT5, SGLT1 and T1R3 taste receptor subunit, in the trachea of rats using immunohistochemistry and immunoelectron microscopy, and compared them using double-labeled confocal microscopy. We found that GLUT2, GLUT5, SGLT1 and T1R3 are selectively expressed in different cell types. T1R3 and GLUT2 are predominantly expressed in subsets of solitary chemoreceptor cells (SCCs) and ciliated cells, GLUT5 is present in subsets of SCCs and in secretory cells, and SGLT1 is exclusively expressed in a unique cell type, SCCs. Furthermore, we demonstrated that T1R3 is colocalized with SGLT1 in SCCs and with GLUT2 transporter in ciliated cells. In conclusion, these findings reveal that different cell types are associated with the uptake of glucose in ASL and that, due to their T1R3 expression, SCCs and ciliated cells are most likely to participate in the chemosensory process in ASL.

Testing homeopathy in mouse emotional response models: pooled data analysis of two series of studies.

Two previous investigations were performed to assess the activity of Gelsemium sempervirens (Gelsemium s.) in mice, using emotional response models. These two series are pooled and analysed here. Gelsemium s. in various homeopathic centesimal dilutions/dynamizations (4C, 5C, 7C, 9C, and 30C), a placebo (solvent vehicle), and the reference drugs diazepam (1 mg/kg body weight) or buspirone (5 mg/kg body weight) were delivered intraperitoneally to groups of albino CD1 mice, and their effects on animal behaviour were assessed by the light-dark (LD) choice test and the open-field (OF) exploration test. Up to 14 separate replications were carried out in fully blind and randomised conditions. Pooled analysis demonstrated highly significant effects of Gelsemium s. 5C, 7C, and 30C on the OF parameter "time spent in central area" and of Gelsemium s. 5C, 9C, and 30C on the LD parameters "time spent in lit area" and "number of light-dark transitions," without any sedative action or adverse effects on locomotion. This pooled data analysis confirms and reinforces the evidence that Gelsemium s. regulates emotional responses and behaviour of laboratory mice in a nonlinear fashion with dilution/dynamization.

Análise de dados com modelos comportamentais em camundongos submetidos a Gelsemium sepervirens e novos resultados para Ignatia amara e Aconitum napellus / Mice behavioural models with pooled data analysis of Gelsemium studies and new findings about Ignatia and Aconitum

Objective: We investigated the effects of three different homeopathic medicines in several dilutions/dynamizations on mice, using validated models which explore anxiety-like and emotional symptoms. Two complete series of investigations were performed in order to assess the activity of Gelsemium sempervirens; furthermore, we investigated Ignatia amara and Aconitum napellus in the same model systems.Conclusions: Pharmacological effects of highly diluted/dynamized medicines on behavioural and emotional symptoms are clearly detectable also in experimental animals. Among the three tested remedies, in these model paradigms Gelsemium showed the highest activity, which was statistically significant as compared with pure solvent even in solutions diluted beyond the Avogadro constant. The effects of different medicines on the anxiety-like symptoms were qualitatively different and differed also from those of benzodiazepines and of serotonin partial agonists. These experiences raise some technical issues related to the animal models and to the possible translation to homeopathy in humans.Acknowledgments: This work was supported by grants from Laboratoires Boiron s.r.l. (Milano, I) to Verona University - Department of Pathology and Diagnostics.(AU)

Objetivo: Nós investigamos os efeitos de três diferentes medicamentos homeopáticos em várias diluições/dinamizações em camundongos, usando modelos validados que exploram sintomas de ansiedade e de emocionalidade. Duas séries completas de investigações foram feitas de forma a estudar a atividade do Gelsemium sempervirens. Além disso, investigamos a Ignatia amara e o Aconitum napellus utilizando os mesmos modelos.Métodos: Camundongos da linhagem CD1 foram randomizados em diferentes gaiolas (mínimo de 8 camundongos por tratamento em cada experimento) e as soluções foram codificadas de forma que todos os protocolos fossem executados completamente em cego. Os compostos indicados em várias diluições/dinamizações centesimais, a solução controle (solvente dinamizado) ou drogas de referência, como o Diazepam (1 mg/kg PV) ou a Buspirona (5 mg/kg PV) diluídos no mesmo solvente sucussionado foram administrados por via ip (0.3 ml/camundongo) por 9 dias. Uma série de mudanças no comportamento dos animais foi monitorada pelo teste de escolha claro/escuro (LD) e pelo campo aberto (OF). Duas séries com pequenas diferenças técnicas, explorando 14 experimentos independentes forem feitas com Gelsemium, cinco experimentos completos foram feitos com Ignatia e quatro com Aconitum.Resultados: Em ambas as séries de experimentos feitos com Gelsemium, observaram-se efeitos de padrão ansiolítico em ambos os testes (OF e LD). Contudo, devido à grande variabilidade das respostas dos animais e possivelmente de algumas pequenas diferenças nos protocolos, esses efeitos alcançaram o limiar de significância estatística apenas para o campo aberto na primeira série e para o teste claro/escuro na segunda série. A análise cumulativa das duas séries demonstrou significância muito alta (p?0,0001) dos efeitos do Gelsemium 5CH, 7CH e 30CH para os parâmetros do campo aberto e do Gelsemium 5CH, 9CH e 30CH para os parâmetros do teste claro/escuro. No campo aberto, o efeito do Gelsemium foi particularmente diferente em comparação com as drogas de referência, sugerindo aumento no comportamento exploratório e redução naInt J High Dilution Res 2011; 10(36):198-200Proceedings of the XXV GIRI Symposium and VIII CBFH; 2011 Sep 04-07; Foz do Iguaçu (Brazil)200tigmotaxia e na neofobia, ao contrário do que se esperaria de um efeito ansiolítico clássico; no teste claro/escuro, os efeitos do Gelsemium foram similares aos do benzodiazepínico. A Ignatia mostrou efeitos estatisticamente significativos apenas no teste claro/escuro (pico na diluição 9CH), enquanto que o Aconitum apresentou efeito similar apenas em um experimento, onde o nível de ansiedade basal dos animais estava previamente elevado. Também observamos uma tendência de Ignatia em diminuir o número de marcas de urina e do Aconitum de diminuir o número de bolos fecais, ambos os efeitos são similares ao esperado para o Diazepam. Os medicamentos homeopáticos não alteraram a locomoção geral dos camundongos no campo aberto, indicando que seus efeitos foram realmente semelhantes ao ansiolítico e não simplesmente sedativos, enquanto que a Buspirona inibiu significativamente este parâmetro.Conclusões: Os efeitos farmacológicos de medicamentos preparados em altas diluições/dinamizações sobre os sintomas comportamentais e emocionais são claramente detectáveis também em modelos animais. Entre os três medicamentos estudados, Gelsemium mostrou a maior atividade, que foi estatisticamente significativa quando comparado com o solvente, mesmo quando preparado acima do número de Avogadro. Os efeitos dos diferentes medicamentos sobre os sintomas semelhantes à ansiedade são qualitativamente diferentes e diferem também dos benzodiazepínicos e dos agonistas parciais da serotonina. Tais experiências permitem o desenvolvimento técnico de modelos animais e possíveis extrapolações para a homeopatia humana.Agradecimentos: Esse trabalho recebeu suporte do Laboratório Boiron s.r.l. (Milão, I) para o departamento de Patologia e Diagnóstico da Universidade de Verona.(AU)

Epithelial membrane transporters expression in the developing to adult mouse vomeronasal organ and olfactory mucosa.

To contribute clarifying mechanisms operating in nose chemosensory epithelia and their developmental patterns, we analyzed the expression of different epithelial membrane transporters as well as the Clara cell secretory protein, CC26 in the olfactory, vomeronasal organ (VNO), and respiratory epithelia of embryonic (E13-E19) and postnatal (P1-P60) mice by means of immunohistochemistry and reverse transcriptase-polymerase chain reaction. Results showed that CC26, cAMP-activated chloride channel (CFTR), and the water channel protein aquaporin 2, 3, 4, and 5 (AQP2, AQP3, AQP4, and AQP5) are expressed in developing to adult chemosensory epithelia with differential timing; moreover, their pattern of expression is not identical in VNO and olfactory epithelia as well as the corresponding associated glands; co-localization experiments using olfactory marker protein showed that CFTR, CC26, and AQP4 are not expressed in olfactory neurones. CFTR is expressed in sustentacular cells of the VNO and olfactory epithelium as well as blood vessels of the underlying mucosa, and VNO (but not Bowman's) glands; a similar pattern (excluding blood vessels) is present for AQP2; AQP4 is found in the two chemosensory epithelia and in Bowman's glands. AQP3 is expressed in the olfactory epithelium and the associated Bowman's glands, but not in the VNO chemosensory epithelium and glands. AQP5 is expressed in the olfactory epithelium and both Bowman's and VNO glands. These results indicate that water/ions handling as well as antioxidant mechanisms operating at the surface and/or inside the nose chemosensory epithelia start developing in utero and are maintained up to sexual maturity.

Glucose transporters are expressed in taste receptor cells.

In the intestine, changes of sugar concentration generated in the lumen during digestion induce adaptive responses of glucose transporters in the epithelium. A close matching between the intestinal expression of glucose transporters and the composition and amount of the diet has been provided by several experiments. Functional evidence has demonstrated that the regulation of glucose transporters into enterocytes is induced by the sensing of sugar of the enteroendocrine cells through activation of sweet taste receptors (T1R2 and T1R3) and their associated elements of G-protein-linked signaling pathways (e.g. α-gustducin, phospholipase C ß type 2 and transient receptor potential channel M5), which are signaling molecules also involved in the perception of sweet substances in the taste receptor cells (TRCs) of the tongue. Considering this phenotypical similarity between the intestinal cells and TRCs, we evaluated whether the TRCs themselves possess proteins of the glucose transport mechanism. Therefore, we investigated the expression of the typical intestinal glucose transporters (i.e. GLUT2, GLUT5 and SGLT1) in rat circumvallate papillae, using immunohistochemistry, double-labeling immunofluorescence, immunoelectron microscopy and reverse transcriptase-polymerase chain reaction analysis. The results showed that GLUT2, GLUT5 and SGLT1 are expressed in TRCs; their immunoreactivity was also observed in cells that displayed staining for α-gustducin and T1R3 receptor. The immunoelectron microscopic results confirmed that GLUT2, GLUT5 and SGLT1 were predominantly expressed in cells with ultrastructural characteristics of chemoreceptor cells. The presence of glucose transporters in TRCs adds a further link between chemosensory information and cellular responses to sweet stimuli that may have important roles in glucose homeostasis, contributing to a better understanding of the pathways implicated in glucose metabolism.

Expression of taste receptors in solitary chemosensory cells of rodent airways.

BACKGROUND: Chemical irritation of airway mucosa elicits a variety of reflex responses such as coughing, apnea, and laryngeal closure. Inhaled irritants can activate either chemosensitive free nerve endings, laryngeal taste buds or solitary chemosensory cells (SCCs). The SCC population lies in the nasal respiratory epithelium, vomeronasal organ, and larynx, as well as deeper in the airway. The objective of this study is to map the distribution of SCCs within the airways and to determine the elements of the chemosensory transduction cascade expressed in these SCCs. METHODS: We utilized a combination of immunohistochemistry and molecular techniques (rtPCR and in situ hybridization) on rats and transgenic mice where the Tas1R3 or TRPM5 promoter drives expression of green fluorescent protein (GFP). RESULTS: Epithelial SCCs specialized for chemoreception are distributed throughout much of the respiratory tree of rodents. These cells express elements of the taste transduction cascade, including Tas1R and Tas2R receptor molecules, α-gustducin, PLCß2 and TrpM5. The Tas2R bitter taste receptors are present throughout the entire respiratory tract. In contrast, the Tas1R sweet/umami taste receptors are expressed by numerous SCCs in the nasal cavity, but decrease in prevalence in the trachea, and are absent in the lower airways. CONCLUSIONS: Elements of the taste transduction cascade including taste receptors are expressed by SCCs distributed throughout the airways. In the nasal cavity, SCCs, expressing Tas1R and Tas2R taste receptors, mediate detection of irritants and foreign substances which trigger trigeminally-mediated protective airway reflexes. Lower in the respiratory tract, similar chemosensory cells are not related to the trigeminal nerve but may still trigger local epithelial responses to irritants. In total, SCCs should be considered chemoreceptor cells that help in preventing damage to the respiratory tract caused by inhaled irritants and pathogens.

Amylase expression in taste receptor cells of rat circumvallate papillae.

The chemical composition of the luminal content is now accepted to have a profound influence on the performance of chemosensory receptors. Gustatory and intestinal chemoreceptors have in common their expression of molecules involved in taste sensing and signal transduction pathways. The recent finding that enterocytes of the duodenal epithelium are capable of expressing luminal pancreatic amylase suggests that taste cells of the gustatory epithelium might, in the same way, express salivary amylase in the oral cavity. Therefore, we investigated amylase expression in rat circumvallate papillae by using analyses involving immunohistochemistry, Western blot, and reverse transcription with the polymerase chain reaction. In addition, we used double-labeling confocal laser microscopy to compare amylase immunolabeling with that of the following markers: protein gene product 9.5 (PGP 9.5) and chromogranin A (CgA) for endocrine cells, alpha-gustducin and phospholipase C beta 2 (PLC beta 2) as taste-signaling molecules, and cystic fibrosis transmembrane regulator (CFTR) and Clara-cell-specific secretory protein of 10-kDa (CC10) as secretory markers. The results showed that amylase was present in some taste bud cells; its immunoreactivity was observed in subsets of cells that expressed CgA, alpha-gustducin, PLC beta 2, CFTR, or CC10. PGP 9.5 immunoreactivity was never colocalized with amylase. The data suggest that amylase-positive cells constitute an additional subset of taste receptor cells also associated with chemoreceptorial and/or secretory molecules, confirming the occurrence of various pathways in taste buds.

Immunohistochemical localization of Clara cell secretory proteins (CC10-CC26) and Annexin-1 protein in rat major salivary glands.

The oral cavity is continuously bathed by saliva secreted by the major and minor salivary glands. Saliva is the first biological medium to confront external materials that are taken into the body as part of food or drink or inhaled volatile substances, and it contributes to the first line of oral defence. In humans, it has been shown that sputum and a variety of biological fluids contain Clara cell secretory proteins (CC10-CC26). Various studies of the respiratory apparatus have suggested their protective effect against inflammatory response and oxidative stress. Recently, CC10 deficiency has been related to the protein Annexin-1 (ANXA1), which has immunomodulatory and anti-inflammatory properties. Considering the defensive role of both Clara cell secretory proteins and ANXA1 in the respiratory apparatus, and the importance of salivary gland secretion in the first line of oral defence, we decided to evaluate the expression of CC10, CC26 and ANXA1 proteins in rat major salivary glands using immunohistochemistry. CC10 expression was found only in the ductal component of the sublingual gland. Parotid and submandibular glands consistently lacked CC10 immunoreactivity. In the parotid gland, both acinar and ductal cells were always CC26-negative, whereas in the submandibular gland, immunostaining was localized in the ductal component and in the periodic acid Schiff (PAS)-positive area. In the sublingual gland, ductal cells were always positive. Acinar cells were not immunostained at all. ANXA1 was expressed in ductal cells in all three major glands. In parotid and sublingual glands, acinar cells were negative. In submandibular glands, immunostaining was present in the mucous PAS-positive portion, whereas serous acinar cells were consistently negative. The existence of some CC10-CC26-ANXA1-positive cells in rat salivary glandular tissue is an interesting preliminary finding which could support the hypothesis, suggested for airway tissue, that these proteins have a defensive and protective role. Protein expression heterogeneity in the different portions of the glands could be an important clue in further investigations of their role.