Hydrogel-based milliwell arrays for standardized and scalable retinal organoid cultures.

The development of improved methods to culture retinal organoids is relevant for the investigation of mechanisms of retinal development under pathophysiological conditions, for screening of neuroprotective compounds, and for providing a cellular source for clinical transplantation. We report a tissue-engineering approach to accelerate and standardize the production of retinal organoids by culturing mouse embryonic stem cells (mESC) in optimal physico-chemical microenvironments. Arrayed round-bottom milliwells composed of biomimetic hydrogels, combined with an optimized medium formulation, promoted the rapid generation of retina-like tissue from mESC aggregates in a highly efficient and stereotypical manner: ∼93% of the aggregates contained retinal organoid structures. 26 day-old retinal organoids were composed of ∼80% of photoreceptors, of which ∼22% are GNAT2-positive cones, an important and rare sensory cell type that is difficult to study in rodent models. The compartmentalization of retinal organoids into predefined locations on a two-dimensional array not only allowed us to derive almost all aggregates into retinal organoids, but also to reliably capture the dynamics of individual organoids, an advantageous requirement for high-throughput experimentation. Our improved retinal organoid culture system should be useful for applications that require scalability and single-organoid traceability.

Obesity candidate genes, gestational weight gain, and body weight changes in pregnant women.

OBJECTIVE: To examine the associations of two obesity-associated genes, FTO (rs9939609) and GNB3 (rs5443) single nucleotide polymorphisms (SNPs), with early pregnancy body mass index, gestational weight gain, and postpartum weight retention. METHODS: Secondary data analysis of self-identified white (n = 580) and black (n = 194) women who participated in a randomized controlled trial (2009-2014) and provided a saliva sample of DNA. Bivariate relationships were assessed using analysis of variance. Multiple regression models assessed the relationship between outcomes and gene SNPs, controlling for income, parity, and smoking status. RESULTS: FTO and GNB3 gene associations with pregnancy weight were different by racial group and early pregnancy body mass index. Obese black women homozygote for the FTO risk allele (AA) had a higher gestational weight gain compared with non-risk homozygotes (TT) (P = 0.006). GNB3 non-risk CC homozygotes tended to have a lower gestational weight gain compared with heterozygotes (P = 0.05). White GNB3 C carriers tended to be heavier in early pregnancy (P <0.1) and GNB3 homozygote (TT) overweight women tended to have lower postpartum weight retention than C carriers. CONCLUSIONS: The FTO gene and possibly the GNB3 gene are associated with high gestational weight gain in obese black women. Obese carriers of the FTO risk allele gained 4.1 kg (AT) and 7.6 kg (TT) more than those without risk alleles. Overweight GNB3 heterozygotes (CT) gained 6.6 kg less than homozygotes (CC). Overweight or obese black women who have either risk variant are at risk for high gestational weight gain.

Papel de las subunidades alfa de proteínas G en los procesos morfogénicos de hongos filamentosos de la división Ascomycota / Role of G-protein alpha sub-units in the morphogenic processes of filamentous Ascomycota fungi

La división Ascomycota comprende alrededor del 75% de las especies fúngicas descritas e incluye especies de enorme importancia médica, fitosanitaria, agrícola y biotecnológica. La capacidad para propagarse, explorar y colonizar nuevos sustratos es una característica de vital importancia para este grupo de organismos. En ese sentido, procesos como la germinación conidial, la extensión de las hifas y la esporulación constituyen el eje central del desarrollo en la mayoría de los hongos filamentosos. Estos procesos requieren de una maquinaria morfogénica especializada, coordinada y regulada por mecanismos que aún están siendo dilucidados. En los últimos años se ha avanzado sustancialmente en la comprensión del papel que desempeña la ruta de señalización mediada por proteínasG heterotriméricas en los procesos biológicos básicos de diversos hongos filamentosos. Por lo anterior, esta revisión se enfoca en el papel que desempeñan las subunidades alfa de dichas proteínas en los procesos morfogénicos de los hongos filamentosos de la división Ascomycota (AU)

The phylum Ascomycota comprises about 75% of all the fungal species described, and includes species of medical, phytosanitary, agricultural, and biotechnological importance. The ability to spread, explore, and colonise new substrates is a feature of critical importance for this group of organisms. In this regard, basic processes such as conidial germination, the extension of hyphae and sporulation, make up the backbone of development in most filamentous fungi. These processes require specialised morphogenic machinery, coordinated and regulated by mechanisms that are still being elucidated. In recent years, substantial progress has been made in understanding the role of the signalling pathway mediated by heterotrimericG proteins in basic biological processes of many filamentous fungi. This review focuses on the role of the alpha subunits of heterotrimericG proteins in the morphogenic processes of filamentous Ascomycota (AU)

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.

mGluR6 deletion renders the TRPM1 channel in retina inactive.

In darkness, glutamate released from photoreceptors activates the metabotropic glutamate receptor 6 (mGluR6) on retinal ON bipolar cells. This activates the G protein G(o), which then closes transient receptor potential melastatin 1 (TRPM1) channels, leading to cells' hyperpolarization. It has been generally assumed that deleting mGluR6 would render the cascade inactive and the ON bipolar cells constitutively depolarized. Here we show that the rod bipolar cells in mGluR6-null mice were hyperpolarized. The slope conductance of the current-voltage curves and the current noise were smaller than in wild type. Furthermore, while in wild-type rod bipolar cells, TRPM1 could be activated by local application of capsaicin; in null cells, it did not. These results suggest that the TRPM1 channel in mGluR6-null rod bipolar cells is inactive. To explore the reason for this lack of activity, we tested if mGluR6 deletion affected expression of cascade components. Immunostaining for G protein subunit candidates Gα(o), Gß(3), and Gγ(13) showed no significant changes in their expression or distribution. Immunostaining for TRPM1 in the dendritic tips was greatly reduced, but the channel was still present in the soma and primary dendrites of mGluR6-null bipolar cells, where a certain fraction of TRPM1 appears to localize to the plasma membrane. Consequently, the lack of TRPM1 activity in the null retina is unlikely to be due to failure of the channels to localize to the plasma membrane. We speculate that, to be constitutively active, TRPM1 channels in ON bipolar cells have to be in a complex, or perhaps require an unidentified factor.

[Functional state of adenylate cyclase signaling system in testis and ovary of fasted rats].

Sensitivity of the adenylate cyclase signaling system (ACSS) to polypeptide hormones and biogenic amines is studied in testis and ovary of rats after the 2- and 4-day fasting as compared with control animals. In tissues of the fasted rats there is shown a decrease in the basal activity of adenylate cyclase (AC) and of the basal level of the GTP binding of heterotrimeric G protein. An increase of duration of fasting from 2 to 4 days led to intensification of these changes. In the fasted rats, the stimulating effects of chorionic gonadotropin, PACAP-38. and isoproterenol on the AC activity realized via G protein of the stimulatory type are enhanced, whereas the inhibitory effects of somatostatin on the AC activity realized via G protein of the inhibitory type are reduced. In testis of the fasted rats the stimulating effect of serotonin acting on AC via both types of G proteins are increased, while the inhibitory effects of the hormone decrease. Thus, under conditions of fasting, in rat testis and ovary the ACSS sensitivity to regulatory effects of hormones is changing: its stimulatory effects are increased, while its inhibitory effects, on the contrary, are decreased. We suggest these changes is one of the key mechanisms of adaptation of organism to deficiency of nutritional resources to be aimed at intensifying the tissues catabolic processes, preferably, lypolysis.

Reduction of type II taste cells correlates with taste dysfunction after X-ray irradiation in mice.

BACKGROUND: Taste dysfunction that develops after radiotherapy for head and neck cancer impairs patients' quality of life. Although taste cells have been shown to degenerate after exposure to X-ray irradiation, the alteration in taste cell population is unclear. This study investigated the histopathological change of taste bud structure and the taste cell population in X-ray irradiated mice. METHODS: The head and neck region of C57BL/6J male mice was exposed to a single 15 Gy dose of X-ray irradiation and a chronological histopathological analysis of the circumvallate papilla was performed. Preference for sweet taste was measured using the two-bottle preference method. RESULTS: The histological analysis of the circumvallate papilla revealed that the basal cells had almost disappeared, but that there was not clear change in the spindle-shaped taste cells on day 4 after irradiation. The number of taste cells had decreased on day 8, and then remained unchanged until day 20, after which they increased and recovered to their original number by day 24. There was a more marked decrease in the number of alpha-gustducin-positive type II taste cells than in the number of serotonin-positive type III taste cells. Preference for sweet taste measured by the two-bottle preference method was decreased in parallel with taste cell number. CONCLUSION: These findings suggest that X-ray irradiation disrupts the basal cells, resulting in a decrease of the number of taste cells, particularly type II taste cells, which may be the cause of radiotherapy-induced taste dysfunction.

Characterization of umami receptor and coupling G protein in mouse taste cells.

Taste receptor cells (TRCs) express multiple umami receptors. We performed physiological investigations to determine whether umami-responding cells in taste buds possess G protein-coupled receptors and to determine what type of G proteins exist if any. To clarify the components that participate in intracellular umami signal transduction in mouse, we recorded the activation of TRCs. TRCs treated with the G protein inhibitor GDP-beta-S lost umami-induced inward currents. Treatment with the Galphai inhibitor, pertussis toxin, did not increase the intracellular Ca2+ level in many TRCs. Immunohistochemical analysis revealed that a subset of TRCs responding to umami stimuli expressed alpha-gustducin. Thus, we demonstrated that umami stimuli were received by G protein-coupled receptors that function together with some of the Galphai family members.

Ric-8B interacts with G alpha olf and G gamma 13 and co-localizes with G alpha olf, G beta 1 and G gamma 13 in the cilia of olfactory sensory neurons.

Olfactory sensory neurons are able to detect odorants with high sensitivity and specificity. We have demonstrated that Ric-8B, a guanine nucleotide exchange factor (GEF), interacts with Galphaolf and enhances odorant receptor signaling. Here we show that Ric-8B also interacts with Ggamma13, a divergent member of the Ggamma subunit family which has been implicated in taste signal transduction, and is abundantly expressed in the cilia of olfactory sensory neurons. We show that Gbeta1 is the predominant Gbeta subunit expressed in the olfactory sensory neurons. Ric-8B and Gbeta1, like Galphaolf and Ggamma13, are enriched in the cilia of olfactory sensory neurons. We also show that Ric-8B interacts with Galphaolf in a nucleotide dependent manner, consistent with the role as a GEF. Our results constitute the first example of a GEF protein that interacts with two different olfactory G protein subunits and further implicate Ric-8B as a regulator of odorant signal transduction.

Measurement of heterotrimeric G-protein and regulators of G-protein signaling interactions by time-resolved fluorescence resonance energy transfer.

G-protein-coupled receptors transduce their signals through G-protein subunits which in turn are subject to modulation by other intracellular proteins such as the regulators of G-protein signaling (RGS) proteins. We have developed a cell-free, homogeneous (mix and read format), time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor heterotrimeric G-protein subunit interactions and the interaction of the G alpha subunit with RGS4. The assay uses a FRET pair consisting of a terbium cryptate chelate donor spectrally matched to an Alexa546 fluor acceptor, each of which is conjugated to separate protein binding partners, these being G alpha(i1):beta4gamma2 or G alpha(i1):RGS4. Under conditions favoring specific binding between labeled partners, high-affinity interactions were observed as a rapid increase (>fivefold) in the FRET signal. The specificity of these interactions was demonstrated using denaturing or competitive conditions which caused significant reductions in fluorescence (50-85%) indicating that labeled proteins were no longer in close proximity. We also report differential binding effects as a result of altered activation state of the G alpha(i1) protein. This assay confirms that interactions between G-protein subunits and RGS4 can be measured using TR-FRET in a cell- and receptor-free environment.

Does mRNA level of microsomal carnitine palmitoyltransferase predict yield of peripheral blood stem cell apheresis?

Transplantation of autologous hematopoietic stem cells is a well established therapeutic procedure. Despite advances in efficacy of the stem cell mobilization and apheresis process until now a predictive factor for the expected stem cell yield before initiation of mobilization therapy could not be identified. The main objective of our study was to evaluate alterations in enzymes involved in fatty acid metabolism on the level of gene expression in mononuclear cells, as changes in relative mRNA levels of these enzymes could represent the hematopoietic regenerative potential. Data of 23 consecutive patients with different lymphoid malignancies undergoing stem cell mobilization were analyzed. Our results show that mRNA levels of microsomal carnitine palmitoyltransferase in peripheral blood mononuclear cells quantified before application of mobilization therapy correlate positively with the amount of CD34 positive cells in peripheral blood before first apheresis, in the first apheresis product and in the total harvest outcome. The association of enzymes involved in fatty acid metabolism with hematoopoiesis was further confirmed in healthy subjects on altitude-adaptation training and in proliferating or differentiating HL60 cells. This gives evidence for a possible predictive value of such analyzes though further data of a larger sample are to be collected to confirm our observations.

Importancia de las proteínas G heterotriméricas en la biología molecular del cáncer de próstata / Importance of heterotrimetric G proteins in prostate cancer molecular biology

Objetivo: Profundizar en el conocimiento de la implicación de las subunidades αs y αi de las proteínas G heterotriméricas en el cáncer prostático humano. Métodos: Tejido prostático de 9 pacientes sometidos a prostatectomía radical por cáncer prostático y 5 controles sometidos a cistoprostatectomía por carcinoma de vejiga. Se analizó la expresión de subunidades αs y αi(mRNA por RT-PCR y proteína por Western-blot), funcionalidad (actividad adenilil ciclasa, AC) y posibilidad de mutaciones (análisis de restricción enzimática y secuenciación del cDNA).Resultados: A nivel de mRNA, se detectó la expresión de subunidades αs, αi1, αi2 y αi3 en tejidos sanos y cancerosos. A nivel de proteína, la expresión de subunidades αs y αi1,2 disminuyó (25% y 40%, respectivamente) en cáncer de próstata. También disminuyó la expresión de αi3,0, mientras que la de ß no se modificó. La actividad basal AC en membranas de adenocarcinoma fue 40% inferior al control. La digestión con enzimas de restricción Eag I o AlwN I no permitió localizar mutaciones en αs. Sin embargo, la digestión a nivel de αi2 con BstU I sirvió para observar en tejido canceroso un cambio de Gln205 (triplete CAG) a Pro (CCG).Conclusiones: La funcionalidad y expresión de las subunidades de las proteínas G heterotriméricas están modificadas selectivamente en el adenocarcinoma prostático humano, concurriendo además alguna mutación puntual. La sustitución observada de Glu205 por Pro puede conllevar que la proteína αi2 resultante tenga muy baja actividad GTPasa y, por tanto, se encuentre estabilizada en su forma activa (AU)

Objective: To deep in the knowledge of the involvement of G-protein αs and αi subunits in human prostate cancer. Methods: Prostate tissue from 9 patients undergoing radical prostatectomy for prostate cancer and 5 controls undergoing cystoprostatectomy for bladder carcinoma. G-protein αs and αi subunits were studied for expression (mRNA by RT-PCR and protein by Western-blot), functionality (adenylyl cyclase activity, AC) and possibility of mutations (analysis with restriction enzymes and cDNA sequentiation). Results: At mRNA level, the expression of αs, αi1, αi2 y αi3 was detected in healthy and cancerous tissues. At protein level, the expression of αs y αi1,2 diminished (25% and 40%, respectively) in prostate cancer. The expression of αi3/0 also diminished, whereas that of ß subunit was not modified. Basal AC activity in adenocarcinoma membranes was 40% inferior to the control. Digestion with restriction enzymes Eag I or AlwN I did not allow to locate mutations in αs. However, digestion at αi2 level with BstU I enzyme served to observe a change of Gln205 (CAG triplet) to Pro (CCG). Conclusions: The functionality and expression of heterotrimeric G proteins are selectively modified in human prostate adenocarcinoma, occurring in addition some punctual mutation. The observed substitution of Gln205 by Pro may result in a low GTPase activity for αi2 that, therefore, is stabilized in its active form (AU)

G protein threshold behavior in the human neutrophil oxidant response: measurement of G proteins available for signaling in responding and nonresponding subpopulations.

Threshold behavior is an important aspect of signal transduction pathways that allows for responses to be turned on or off. Human neutrophil responses to N-formyl peptides, including oxidant production and release, exhibit threshold behavior with respect to the number of G proteins available for signaling; progressive treatment of neutrophils with pertussis toxin causes the conversion of responding cells to nonresponding cells. To quantify the threshold level of G proteins required for signaling of N-formyl peptide stimulated oxidant production in a neutrophil population, we used a plasma membrane associated G protein quantification assay in conjunction with a sorting flow cytometer and measured differences in the average number of G proteins available for signaling per cell in both the responding and the nonresponding subpopulations after pertussis toxin treatment. Although there appeared to be a threshold separating responding cells and nonresponding cells for a given sample, no discrete threshold was measured across multiple treatment conditions. A mathematical model of the early steps in signaling suggests that cell-to-cell variability in signal parameters, such as numbers of signal components and values of kinetic rate constants, obscures the measurement of a discrete threshold and leads to an apparent decrease in the threshold level of G proteins available for signaling as the total G proteins are decreased.

Heterotrimeric G protein subunits are located on rat liver endosomes.

BACKGROUND: Rat liver endosomes contain activated insulin receptors and downstream signal transduction molecules. We undertook these studies to determine whether endosomes also contain heterotrimeric G proteins that may be involved in signal transduction from G protein-coupled receptors. RESULTS: By Western blotting Gsalpha, Gialpha1,2, Gialpha3 and Gbeta were enriched in both canalicular (CM) and basolateral (BLM) membranes but also readily detectable on three types of purified rat liver endosomes in the order recycling receptor compartment (RRC) > compartment for uncoupling of receptor and ligand (CURL) > multivesicular bodies (MVB) >> purified secondary lysosomes. Western blotting with antibodies to Na, K-ATPase and to other proteins associated with plasma membranes and intracellular organelles indicated this was not due to contamination of endosome preparations by CM or BLM. Adenylate cyclase (AC) was also identified on purified CM, BLM, RRC, CURL and MVB. Percoll gradient fractionation of liver postnuclear supernatants demonstrated co-occurrence of endosomes and heterotrimeric G protein subunits in fractions with little plasma membrane markers. By confocal microscopy, punctate staining for Gsalpha, Gialpha3 and Gbeta corresponded to punctate areas of endocytosed Texas red-dextran in hepatocytes from control and cholera toxin-treated livers. CONCLUSION: We conclude that heterotrimeric G protein subunits as well as AC likely traffic into hepatocytes on endosome membranes, possibly generating downstream signals spatially separate from signalling generated at the plasma membrane, analogous to the role(s) of internalized insulin receptors.

Enhanced G(i) signaling selectively negates beta2-adrenergic receptor (AR)--but not beta1-AR-mediated positive inotropic effect in myocytes from failing rat hearts.

BACKGROUND: Myocardial contractile response to beta1- and beta2-adrenergic receptor (AR) stimulation is severely impaired in chronic heart failure, in which G(i) signaling and the ratio of beta2/beta1 are often increased. Because beta2-AR but not beta1-AR couples to G(s) and G(i) with the G(i) coupling negating the G(s)-mediated contractile response, we determined whether the heart failure-associated augmentation of G(i) signaling contributes differentially to the defects of these beta-AR subtypes and, if so, whether inhibition of G(i) or selective activation of beta2-AR/G(s) by ligands restores beta2-AR contractile response in the failing heart. METHODS AND RESULTS: Cardiomyocytes were isolated from 18- to 24-month-old failing spontaneously hypertensive (SHR) or age-matched Wistar-Kyoto (WKY) rat hearts. In SHR cardiomyocytes, either beta-AR subtype-mediated inotropic effect was markedly diminished, whereas G(i) proteins and the beta2/beta1 ratio were increased. Disruption of G(i) signaling by pertussis toxin (PTX) enabled beta2- but not beta1-AR to induce a full positive inotropic response in SHR myocytes. Furthermore, screening of a panel of beta2-AR ligands revealed that the contractile response mediated by most beta2-AR agonists, including zinterol, salbutamol, and procaterol, was potentiated by PTX, indicating concurrent G(s) and G(i) activation. In contrast, fenoterol, another beta2-AR agonist, induced a full positive inotropic effect in SHR myocytes even in the absence of PTX. CONCLUSIONS: We conclude that enhanced G(i) signaling is selectively involved in the dysfunction of beta2- but not beta1-AR in failing SHR hearts and that disruption of G(i) signaling by PTX or selective activation of beta2-AR/G(s) signaling by fenoterol restores the blunted beta2-AR contractile response in the failing heart.

Quantitative reverse transcriptase polymerase chain reaction analysis for KiSS-1 and orphan G-protein-coupled receptor (hOT7T175) gene expression in hepatocellular carcinoma.

PURPOSE: KiSS-1 has been cloned as a human metastasis suppressor gene and an orphan G-protein-coupled receptor (hOT7T175) identified as the endogenous receptor of the KiSS-1 product. In the present study, we evaluated the clinical importance of KiSS-1 and hOT7T175 gene expression in hepatocellular carcinoma (HCC). METHODS: The expression levels of KiSS-1, hOT7T175 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNAs (mRNAs) were analyzed quantitatively by real-time reverse transcriptase polymerase chain reaction (RT-PCR) in 60 surgically resected HCCs. The KiSS-1/GAPDH and hOT7T175/GAPDH ratios of tumors were compared with clinicopathological findings. RESULTS: Loss of KiSS-1 mRNA expression was not detected in HCCs. The mean KiSS-1/GAPDH ratio did not change between non-cancerous cirrhotic livers and carcinomas. On the other hand, the average hOT7T175/GAPDH ratios increased from non-cancerous livers (0.08) to carcinomas (0.48). Overexpression of KiSS-1 and hOT7T175 genes was recognized in 6 tumors, which were in an advanced stage and showed poor survival. CONCLUSION: Overexpression of KiSS-1 and hOT7T175 genes was frequently observed and correlated with HCC progression; thus, the possibility that overexpressed KiSS-1 and hOT7T175 peptides mediate growth signals into cancer cells in HCCs is suggested.

A functional assay for G-protein-coupled receptors using stably transformed insect tissue culture cell lines.

Insect cells are an underexplored resource for functional G-protein-coupled receptor (GPCR) assays, despite a strong record in biochemical (binding) assays. Here we describe the use of vectors capable of creating stably transformed insect cell lines to generate a cell-based functional GPCR assay. This assay employs the luminescent photoprotein aequorin and the promiscuous G-protein subunit Galpha16 and is broadly applicable to human GPCRs. We demonstrate that the assay can quantitate ligand concentration-activity relationships for seven different human GPCRs, can differentiate between partial and full agonists, and can determine rank order potencies for both agonists and antagonists that match those seen with other assay systems. Human Galpha16 improves signal strength but is not required for activity with some receptors. The coexpression of human and bovine betagamma subunits and/or phospholipase Cbeta makes no difference to agonist efficacy or potency. Two different receptors expressed in the same cell line respond to their specific agonists, and two different cell lines (Sf9 and High 5) are able to functionally detect the same expressed GPCR. Sf9 cells have the capability to produce fully functional human receptors, allied to a low background of endogenous receptors, and so are a valuable system for investigating orphan GPCRs and receptor dimerization.

Immunohistochemistry of the canine vomeronasal organ.

The canine's olfactory acuity is legendary, but neither its main olfactory system nor its vomeronasal system has been described in much detail. We used immunohistochemistry on paraffin-embedded sections of male and female adult dog vomeronasal organ (VNO) to characterize the expression of proteins known to be expressed in the VNO of several other mammals. Basal cell bodies were more apparent in each section than in rodent VNO and expressed immunoreactivity to anticytokeratin and antiepidermal growth factor receptor antibodies. The thin layer of neurone cell bodies in the sensory epithelium and axon fascicles in the lamina propria expressed immunoreactivity to neurone cell adhesion molecule, neurone-specific beta tubulin and protein gene product 9.5. Some neurones expressed growth-associated protein 43 (GAP43): and a number of those also expressed neurone-specific beta tubulin-immunoreactivity. Some axon fascicles were double labelled for those two proteins. The G-protein alpha subunits Gi and Go, involved in the signal transduction pathway, showed immunoreactivity in the sensory cell layer. Our results demonstrate that the canine vomeronasal organ contains a population of cells that expresses several neuronal markers. Furthermore, GAP43 immunoreactivity suggests that the sensory epithelium is neurogenic in adult dogs.

Age-related decreases in the response of aquaporin-5 to acetylcholine in rat parotid glands.

Aquaporin-5 (AQP5) is important in salivary fluid secretion in response to cholinergic and adrenergic stimuli in rat parotid glands. We hypothesized that expression and function of AQP5 might change with age. Acetylcholine and epinephrine induced increases in AQP5 levels in the apical plasma membranes of both young adult and senescent rats. The stimulatory effect of acetylcholine, but not that of epinephrine, on AQP5 levels in the apical plasma membranes of the cells decreased markedly during aging. The quinuclidine derivative, SNI-2011, induced a persistent increase in AQP5 levels in the apical plasma membrane in the cells of both these rats. The amounts of M(3)-muscarinic receptor and Gq proteins did not decrease during aging. The age-related alteration in the responsiveness of AQP5 in the cells to these stimuli might account for the concomitant changes in nitric oxide synthase activity. These results suggest that SNI-2011 might have therapeutic benefit for the treatment of age-related xerostomia.