Expression of Na(+)/K(+)-ATPase alpha subunit isoforms in rat salivary glands: occurrence of sense and antisense RNAs of the alpha3 isoform in the sublingual gland.

We examined the expression of Na(+)/K(+)-ATPase alpha-subunit isoforms in rat salivary glands by RT-PCR. Isoform alpha1 was expressed strongly in all three major salivary glands. The alpha2 isoform was expressed in both submandibular gland (SMG) and sublingual gland (SLG) but faintly in the parotid gland (PG). The alpha3 was detected only in the SLG, and the alpha3 mRNA in the SLG was 1/8 of its level in the brain. Na(+)/K(+)-ATPase alpha3 isoform in the SLG, was localized predominantly on the basolateral plasma membranes in serous cells by immunohistochemical method. We also found the presence of natural antisense RNA of the alpha3 isoform in rat SLG: the 1st-strand cDNA prepared with gene-specific forward primers targeted to the CDS region and to the promoter region of the alpha3 gene in place of oligo(dT) or gene-specific reverse primers produced reasonable PCR products corresponding to the alpha3 cDNA sequence by the subsequent PCR reaction. Synthesis of the 1st-strand cDNA with the gene-specific forward primers was prevented by RNase digestion of the total RNA preparation, indicating that the PCR products in the RT-PCR system were not due to the contaminated genomic DNA, if any. The alpha3 protein level in rat SLG increased with aging, and levels of both alpha3 mRNA (sense RNA) and alpha3 antisense RNA were higher in SLGs of aged rats than in those of young rats, respectively.

Comparative cytotoxicity and ROS generation by curcumin and tetrahydrocurcumin following visible-light irradiation or treatment with horseradish peroxidase.

In order to clarify the cytotoxic mechanism of curcumin, a well-known chemopreventive agent, the cytotoxicity (by MTT method), intracellular glutathione (using GSH detection kit) and intracellular reactive oxygen species (ROS) levels (with a flow cytometer), were measured in curcumin- and tetrahydrocurcumin (TH-curcumin)-treated cancer (HSG) and normal (HGF) cells under two different oxidation conditions: irradiation with visible light (VL) and enzymatic oxidation with horseradish peroxidase (HRP)/H2O2. The cytotoxicity of curcumin was highly enhanced by VL-irradiation, whereas that of TH-curcumin was enhanced by HRP/H2O2 treatment. The cytotoxicity of curcumin against HGF cells was greater than that against HSG cells. Curcumin significantly reduced the intracellular GSH level significantly under VL-irradiation, and increased it under HRP/H2O2, whereas TH-curcumin had no effect with either oxidation treatment. HRP/H2O2 treatment of TH-curcumin enhanced generation of ROS; in contrast, VL-irradiation of curcumin was considered to produce ROS preferably. In conclusion, curcumin was highly photo-toxic, caused a decrease in GSH and mediated ROS generation. In contrast, the cytotoxicity of TH-curcumin was enhanced by enzymatic oxidation. A low-level pro-oxidant intracellular milieu induced by TH-curcumin could be effectively useful for cancer prevention.

Smelling lavender and rosemary increases free radical scavenging activity and decreases cortisol level in saliva.

Free radicals/reactive oxygen species are related to many biological phenomena such as inflammation, aging, and carcinogenesis. The body possesses various antioxidative systems (free radical scavenging activity, FRSA) for preventing oxidative stress, and saliva contains such activity. In the present study, we measured the total salivary FRSA induced after the smelling of lavender and rosemary essential oils that are widely used in aromatherapy. Various physiologically active substances in saliva such as cortisol, secretory IgA, and alpha-amylase activity were found to be correlated with aroma-induced FRSA. The subjects (22 healthy volunteers) sniffed aroma for 5 min, and each subject's saliva was collected immediately. FRSA was measured using 1,1-diphenyl-2-picrylhydrazyl. The FRSA values were increased by stimulation with low concentrations (1000 times dilution) of lavender or by high-concentrations (10 times dilution) of rosemary. In contrast, both lavender and rosemary stimulations decreased cortisol levels. A significant inverse correlation was observed between the FRSA values and the cortisol levels with each concentration of rosemary stimulation. No significant changes were noted in sIgA or alpha-amylase. These findings clarify that lavender and rosemary enhance FRSA and decrease the stress hormone, cortisol, which protects the body from oxidative stress.

Relationships between insulin release and taste.

Tasting sweet food elicits insulin release prior to increasing plasma glucose levels, known as cephalic phase insulin release (CPIR). The characteristic of CPIR is that plasma insulin secretion occurs before the rise of the plasma glucose level. In this experiment, we examined whether taste stimuli placed on the tongue could induce CPIR. We used female Wistar rats and five basic taste stimuli: sucrose (sweet), sodium chloride (salty), HCl (sour), quinine (bitter) or monosodium glutamate (umami). Rats reliably exhibited CPIR to sucrose. Sodium chloride, HCl, quinine, or monosodium glutamate did not elicit CPIR. The non-nutritive sweetener saccharine elicited CPIR. However, starch, which is nutritive but non-sweet, did not elicit CPIR although rats showed a strong preference for starch which is a source of glucose. In addition, we studied whether CPIR was related to taste receptor cell activity. We carried out the experiment in rats with bilaterally cut chorda tympani nerves, one of the gustatory nerves. After sectioning, CPIR was not observed for sweet stimulation. From these results, we conclude that sweetness information conducted by thistaste nerve provides essential information for eliciting CPIR.

Induction of early apoptosis and ROS-generation activity in human gingival fibroblasts (HGF) and human submandibular gland carcinoma (HSG) cells treated with curcumin.

OBJECTIVE: Curcumin [1] is well known to possess apoptosis-inducing activity in some cancer cells, but little is known about its activity in normal cells of oral origin, such as HGF. The aim of the present study was to clarify the relationship between early apoptosis in HGF and the induction of reactive oxygen species (ROS) generation by curcumin. DESIGN: We treated HGF and HSG cells with curcumin [1] and the related compounds biseugenol [2], eugenol [3], alpha-diisoeugenol [4], and isoeugenol [5] and measured cell survival (MTT method), ROS generation (DCFH-DA staining), and induction of early apoptosis. Early apoptosis was detected by monitoring loss of mitochondrial membrane potential (DeltaPsi(m)) by JC-1 staining and externalization of phosphatidylserine (PS) on the cell surface by annexin V-FITC/PI staining combined with flow cytometry. RESULTS: The cytotoxic activities of curcumin [1] and [4] were similar and were nearly 10- to 100-fold stronger than those of the other compounds. Only curcumin was able to induce ROS generation and early apoptosis. Loss of DeltaPsi(m), PS externalization and ROS generation were significantly more pronounced in HGF cells than in HSG cells at curcumin concentrations lower than about 15microM, and were inhibited by the addition of the antioxidants N-acetyl-l-cysteine and glutathione. CONCLUSION: The potent PS externalization and loss of DeltaPsi(m) in curcumin-treated HGF cells appears to be mediated by ROS generation.

Induction of cytotoxicity and apoptosis and inhibition of cyclooxygenase-2 gene expression, by curcumin and its analog, alpha-diisoeugenol.

Cytotoxici and alpha-diisoeugenol were investigated. The cytotoxicity of curcumin and a-diisoeugenol against human promyelocytic leukemia cells (HL-60 cells) and human submandibular cancer cells (HSG cells) was similar (CC50 1-3 microM). However, curcumin induced much more apoptosis, particularly in HL-60 cells compared with HSG cells, as revealed by measurement of the sub-G1/G0 DNA fraction in flow cytometric histograms. Treatment with 15 microM curcumin increased the number of cells with a sub-G1/G0 DNA fraction from control levels of <5% to 55% in HL-60 cells and 30% in HSG cells. Flow cytometry, after staining with annexin V-FITC/PI (the exposure of phosphatidylserine (PS) on the surface of apoptotic cells), showed a dose-dependent induction of early apoptosis by curcumin, which reached about 65% in HL-60 cells and about 20% in HSG cells after treatment with 10 microM curcumin. In contrast, alpha-diisoeugenol failed to induce apoptosis in either cell type. For both cell types, the proportion of late apoptotic/necrotic cells increased rapidly at concentrations of curcumin and a-diisoeugenol greater than 10 microM. The generation of intracellular reactive oxygen species (ROS) in curcumin-treated HL-60 cells was greater than that in HSG cells, as judged by CDFH-DA staining. In both cell types, ROS generation by a-diisoeugenol was at control levels. ROS generation by curcumin was suppressed by antioxidants such as N-acetyl-L-cysteine (NAC) and glutathione (GSH) and by scavengers of hydroxy radicals such as mannitol, but, conversely, was promoted by prooxidants such as the transition metal ions Cu(II) and Zn(II). ROS generation may play a part in the exposure of PS. Curcumin, but not a-diisoeugenol, at 10 microM inhibited LPS (lipopolysaccharide)-induced COX-2 gene expression in RAW 264.7 cells. Semiempirical PM 3 calculations suggested that this activity of curcumin, in which it behaves as a non-steroidal anti-inflammatory drug (NSAID)-like compound, is dependent on its phenolic function, which is more pronounced than that of alpha-diisoeugenol. Taken together, our results suggest that the bioactivity of curcumin is a result of its ability to act as both a prooxidant and an antioxidant.

Evidence for involvement of glucocorticoid response in the hippocampal changes in aged molarless SAMP8 mice.

The involvement of glucocorticoid response in the hippocampal changes in aged SAMP8 mice after removal of their upper molar teeth (molarless condition) was examined using biochemical, morphological and behavioral techniques. Molarless mice showed plasma corticosterone levels to be significantly greater than those in molar-intact control mice. Pretreatment with metyrapone, which suppresses the stress-induced rise in plasma corticosterone levels, prevented the molarless condition-induced increase in plasma corticosterone levels, reduction in CA1 pyramidal neuron numbers, and impairment of spatial learning. The results suggest a link between the molarless condition and the glucocorticoid response, which may be involved in spatial learning deficits and hippocampal neuronal death in aged SAMP8 mice.

The molarless condition in aged SAMP8 mice attenuates hippocampal Fos induction linked to water maze performance.

The involvement of dysfunctional teeth in senile hippocampal activity was evaluated by examining, in aged SAMP8 mice, the effect of cutting off the upper molars (molarless condition) on hippocampal induction of the protein product, Fos, of the immediate early gene, c-fos, and on spatial performance in a water maze. The molarless condition caused a reduction in the number of Fos-positive cells in the hippocampal CA1 region, in which Fos immunoreactivity was localized in the cell nuclei. This effect was more pronounced the longer the molarless condition persisted. The suppression of both learning ability and Fos induction in the CA1 induced by the molarless condition was considerably reduced by restoring the lost molars with artificial crowns. Taken together with the plethora of research showing a relationship between stress, aging and hippocampal function and our past findings [Brain Res. 1999; 826: 148-53; Behav. Brain Res. 2000;108: 145-55; Exp. Gerontol. 2001; 36:283-95], the present results suggest the detrimental effects of a reduction in chewing on hippocampal processing in aged SAMP8 mice that would be linked with stress induced by the molarless condition.

Accessory olfactory bulb neurons are required for maintenance but not induction of V2R vomeronasal receptor gene expression in vitro.

Many mammals detect pheromones by a sensory organ, the vomeronasal organ (VNO). In a previous study using immunoblot and immunocytochemical analyses, we reported that cocultures of VNOs with accessory olfactory bulb (AOB) neurons resulted in the maturation of vomeronasal sensory neurons (VSNs) and a greater expression of V2R family vomeronasal receptors than cultures with VNO alone. To further characterize the V2R expression, we here investigated the time course of the expression of V2R mRNA in the presence or absence of AOB neurons using RT-PCR analysis. The expression of V2R mRNA was already detectable not only in the VNO cocultured with AOB neurons for 3 days in coculture but also in the VNO cultured alone for the same number of days. However, the expression of V2R mRNA in the VNO cultured alone was remarkably decreased during the additional culture period, although that in the cocultured VNO showed sustained expression. Moreover, the application of 2 µM TTX to the cocultured VNO resulted in a marked decrease in the V2R mRNA expression to a level equal to that in the VNO cultured alone for 14 days in coculture. Our previous working hypothesis was that the expression of V2Rs in VSNs was induced by interacting with AOB neurons. However, the present results suggest that the receptor expression in VSNs is independent of the interaction with AOB neurons in the early developmental stage, but is maintained by the active interaction with AOB neurons.

Ameliorative effects of Eriobotrya japonica seed extract on cellular aging in cultured rat fibroblasts.

To investigate the effects of Eriobotrya japonica seed extract (ESE) on cellular aging, intracellular calcium homeostasis in young and senescent cells was analyzed using a rat fibroblast culture as an in vitro model system and a calcium imaging technique. The application of bradykinin (BK) transiently elicited intracellular calcium ion (Ca(2+)) increased in most of the young fibroblasts, whereas these responses were scarcely observed or were significantly attenuated in senescent cells. However, the long-term treatment of senescent cells with ESE (for 7 days) dose-dependently increased the amplitude of BK-induced responses and the percentage of BK-responding cells. In particular, most senescent cells could respond to BK with long-term treatment with ESE (1.0% or 2.0%), an effect that reinstated the percentage of BK-responding cells to the same level as that in young cells. The effects of ESE on amplitude or percentage of responding cells were not observed in young cells. Moreover, the time to half decay, which was significantly longer in senescent cells than that in young cells, was shortened in senescent cells with long-term treatment with ESE. These results suggest that treatment with an adequate concentration of ESE renders BK-induced Ca(2+) dynamics in senescent cells similar to those in young cells. Therefore, ESE can retard and/or protect against cellular aging and may be useful for elucidating the antiaging processes.

Enhanced responses of the chorda tympani nerve to sugars in the ventromedial hypothalamic obese rat.

Sweet taste sensitivity in obese rats with lesions of the ventromedial hypothalamus (VMH) was studied by examining chorda tympani nerve responses to various taste stimuli including sugars. In the early progressive phase of obesity (2 wk after creating VMH lesions), there was no significant difference in the nerve responses to any taste stimulus between sham-operated and VMH-lesioned rats. In contrast, in the late phase of obesity (15-18 wk after VMH lesions), the magnitude of responses to sugars (except for fructose) was prominently greater than that in age-matched controls. High-fat diet-induced obese rats and streptozotocin-diabetic rats also showed greater chorda tympani nerve responses to sugars as was observed in VMH-lesioned obese rats, indicating that VMH lesions might not be specifically related to the enhanced gustatory neural responses to sugars. Although it has been demonstrated that the enhanced responses of the chorda tympani nerve to sugars in genetically diabetic db/db mice is largely attributable to the lack of the direct suppressive effect of leptin on the taste receptor cells, plasma leptin levels were not correlated with the changes in chorda tympani responsiveness to sugars in these models of obesity and diabetes. Accordingly, our results suggest that some chronic factors, including high blood glucose, inefficiency of insulin action, or leptin resistance may be related to the enhancement of chorda tympani nerve responses to sugars.