Relationships between feeding behaviors and emotions: An electroencephalogram (EEG) frequency analysis study

Feeding behaviors may be easily affected by emotions, both being based on brain activity; however, the relationships between them have not been explicitly defined. In this study, we investigated how emotional environments modulate subjective feelings, brain activity, and feeding behaviors. Electroencephalogram (EEG) recordings were obtained from healthy participants in conditions of virtual comfortable space (CS) and uncomfortable space (UCS) while eating chocolate, and the times required for eating it were measured. We found that the more participants tended to feel comfortable under the CS, the more it took time to eat in the UCS. However, the EEG emergence patterns in the two virtual spaces varied across the individuals. Upon focusing on the theta and low-beta bands, the strength of the mental condition and eating times were found to be guided by these frequency bands. The results determined that the theta and low-beta bands are likely important and relevant waves for feeding behaviors under emotional circumstances, following alterations in mental conditions.


Antimony, a novel nerve poison, triggers neuronal autophagic death via reactive oxygen species-mediated inhibition of the protein kinase B/mammalian target of rapamycin pathway.

Antimony (Sb), a naturally occurring metal present in air and drinking water, has been found in the human brain, and there is evidence of its toxic effects on neurobehavioral perturbations, suggesting that Sb is a potential nerve poison. Here, we provide the first study on the molecular mechanism underlying Sb-associated neurotoxicity. Mice exposed to antimony potassium tartrate hydrate showed significantly increased neuronal apoptosis. In vitro, Sb triggered apoptosis in PC12 cells in a dose-dependent manner. Mechanically, Sb triggered autophagy as indicated by increased expression of microtubule-associated protein 1 light chain 3-II (LC3-II) and accumulation of green fluorescent protein-tagged LC3 dots. Moreover, Sb enhanced autophagic flux and sequestosome 1 (p62) degradation. Subsequent analyses showed that Sb treatment decreased phosphorylation of protein kinase B (Akt) as well as the mammalian target of rapamycin (mTOR), while an Akt activator protected PC12 cells from autophagy. Moreover, the antioxidant N-acetylcysteine attenuated Sb-induced Akt/mTOR inhibition and decreased autophagy and apoptosis, with autophagy inhibition also playing a cytoprotective role. In vivo, mice treated with Sb showed higher expression of LC3-II and p62 in the brain, consistent with the in vitro results. In summary, Sb induced autophagic cell death through reactive oxygen species-mediated inhibition of the Akt/mTOR pathway.

The International Journal of Biochemistry & Cell Biology


Interplay between non-NMDA and NMDA receptor activation during oscillatory wave propagation: Analyses of caffeine-induced oscillations in the visual cortex of rats

Generation and propagation of oscillatory activities in cortical networks are important features of the brain. However, many issues related to oscillatory phenomena are unclear. We previously reported neocortical oscillation following caffeine treatment of rat brain slices. Input to the primary visual cortex (Oc1) generates N-methyl-d-aspartate (NMDA) receptor-dependent oscillations, and we proposed that the oscillatory signals originate in the secondary visual cortex (Oc2). Because non-NMDA and NMDA receptors cooperate in synaptic transmission, non-NMDA receptors may also play an important role in oscillatory activities. Here we investigated how non-NMDA receptor activities contribute to NMDA receptor-dependent oscillations by using optical recording methods. After induction of stable oscillations with caffeine application, blockade of NMDA receptors abolished the late stable oscillatory phase, but elicited 'hidden' non-NMDA receptor-dependent oscillation during the early depolarizing phase. An interesting finding is that the origin of the non-NMDA receptor-dependent oscillation moved from the Oc1, during the early phase, toward the origin of the NMDA receptor-dependent oscillation that is fixed in the Oc2. In addition, the frequency of the non-NMDA receptor-dependent oscillation was higher than that of the NMDA receptor-dependent oscillation. Thus, in one course of spatiotemporal oscillatory activities, the relative balance in receptor activities between non-NMDA and NMDA receptors gradually changes, and this may be due to the different kinetics of the two receptor types. These results suggest that interplay between the two receptor types in the areas of Oc1 and Oc2 may play an important role in oscillatory signal communication.


Influences of oral impairment on neural oscilltion and wave propagation in the neocortex of rats


Effects of isoproterenol on aquaporin 5 levels in the parotid gland of mice in vivo.

In the membrane fraction of mouse parotid gland (PG), the protein level of aquaporin 5 (AQP5), a member of the water channel family, was increased by injection (ip) of isoproterenol (IPR), a β-adrenergic agonist, at 1 h, and stayed at high levels until 6 h; this change occurred simultaneously as amylase secretion. The AQP5 level then decreased and returned toward the original level at 12-48 h. After IPR injection, the AQP5 mRNA gradually increased and reached a maximum at 24 h. The facts suggest a rapid appearance of AQP5 at plasma membrane by IPR and subsequent degradation/metabolism by activation of proteolytic systems. Pretreatment of animals with two calpain inhibitors, N-Ac-Leu-Leu-methininal (ALLM) and calpeptin, as well as a protein synthesis inhibitor, cycloheximide (CHX), significantly suppressed the IPR-induced AQP5 degradation in the PG membrane fraction; such suppression was not observed by two proteasome inhibitors, MG132 and lactacystin, or the lysosome denaturant chloroquine, although most of these inhibitors increased AQP5 protein levels in unstimulated mice. The AQP5 protein was also degraded by μ-calpain in vitro. Furthermore, we demonstrated that μ-calpain was colocalized with AQP5 in the acinar cells by immunohistochemistry, and its activity in the PG was increased at 6 h after IPR injection. These results suggest that the calpain system was responsible for IPR-induced AQP5 degradation in the parotid gland and that such a system was coupled to the secretory-restoration cycle of amylase in the PG.

American Journal of Physiology, Endocrinology and Metabolism


Age-dependent emergence of caffeine-assisted voltage oscillations in the endopiriform nucleus of rats.

The gustatory insular cortex (IC) is connected with not only the somatosensory cortex, but also the endopiriform nucleus (EPN). We have previously revealed that low-frequency electrical stimulation to the IC can elicit membrane potential oscillations at a frequency of 8-10 Hz in the somatosensory cortex of rat brain slices under bath-application of caffeine. Using the same procedure, we investigated whether the EPN has the ability to generate oscillations, and whether such oscillations emerge age-dependently. Electrical stimulations were delivered to the IC, and field potentials were recorded from the EPN. In the case of slices made from mature rats, stable field potential oscillations at 8-10 Hz were induced in the EPN after repetitive stimulations. Optical recordings revealed that signals traveled from the IC to the EPN by way of the claustrum. Generation of oscillations was N-methyl-d-aspartate (NMDA) receptor activity-dependent, since oscillatory phases disappeared following application of NMDA receptor antagonist. In slices from immature rats, however, oscillations were not induced. IC stimulation can thus age-dependently elicit membrane potential oscillations in the EPN, and the EPN oscillations were NMDA receptor activity-dependent. These findings suggest that developmental changes in properties of the EPN might contribute to development of information integration, including gustatory information.


Induction of Sca-1 via activation of STAT3 system in the duct cells of the mouse submandibular gland by ligation of the main excretory duct

To examine the very initial step that takes place immediately after tissue injury and is linked to tissue regeneration, we employed the submandibular gland (SMG), which was injured by ligation of its main excretory duct (MED). Ligation of the MED of the SMG in mice induced the expression of Sca-1, a protein marker of hematopoietic stem cells. In the normal gland, a low level of Sca-1 was expressed, which was localized predominantly in the excretory duct cells. At 1 day after ligation, Sca-1 expression increased prominently in almost all of cells in the duct system, but not in the acinar cells. The level of Sca-1 mRNA had begun to increase at 6 h after ligation and continuously rose thereafter until it reached a plateau, which occurred ∼12 h after ligation. STAT3 phosphorylated at its tyrosine-705 (p-STAT3) in the ligated gland increased immediately after ligation, and it was localized in the nuclei of all duct cells. The results of an EMSA revealed the specific binding of a nuclear extract to the sequence of the γ-interferon activation site (GAS) present in the Sca-1 promoter and confirmed that such binding increased after ligation. Thus the present study suggests that STAT3, having been phosphorylated following MED ligation, was transferred to the nucleus, where it bound to the GAS element in the promoter of Sca-1 gene, resulting in promotion of Sca-1 gene expression. Actual prevention of STAT3 phosphorylation reduced the ligation-induced Sca-1 elevation.

American Journal of Physiology, Gastrointestinal and Liver Physiology


Lipopolysaccharide-mediated induction of calprotectin in the submandibular and parotid glands of mice

S100A8 and S100A9 constitute a heterodimeric protein, calprotectin. The mRNAs of S100A8 and S100A9, being expressed at minimal levels in the submandibular and parotid glands (SMG and PG, respectively) of C3H/HeN mice, were induced strongly and transiently by lipopolysaccharide (LPS). Among the mRNAs of members of the S100 protein family examined, those of S100A8 and S100A9 were specifically induced by LPS in the salivary glands. The induction was assumed to be mediated via toll-like receptor 4 (TLR4), since their elevation was limited in C3H/HeJ mice, a TLR4-mutant strain. These proteins became expressed in the granular convoluted tubular cells and striated duct cells in the SMG, and in both acinar and duct cells in the PG (all in the cytoplasm). The salivary calprotectin level was not increased by LPS treatment, implying that elevated calprotectin was not secreted into the saliva and that they may function in microcellular environment of the salivary gland.


Novel phosphorylation of aquaporin-5 at its threonine 259 through cAMP signaling

Aquaporin-5 (AQP5), a water channel, plays key roles in salivary secretion. The novel phosphorylation of AQP5 was investigated by using human salivary gland (HSG) cells and mouse salivary glands. In the HSG cells stably transfected with a wild-type mouse AQP5 construct, a protein band immunoreactive with antibody against phosphorylated PKA substrate was detected in the AQP5 immunoprecipitated sample, and its intensity was enhanced by short-term treatment of the cells with 8-bromo-cAMP, forskolin, or phorbol 12-myristate 13-acetate, but not by that with A23187 calcium ionophore. Such enhancement was inhibited in the presence of H-89, a PKA inhibitor. An AQP5 mutant (AQP5-T259A) expressed by transfection of HSG cells was not recognized by anti-phosphorylated PKA substrate antibody, even when the cells were stimulated with the protein kinase activators. Immunoblotting and immunofluorescence studies using a specific antibody detecting AQP5 phosphorylated at its Thr259 demonstrated that AQP5 was rapidly and transiently phosphorylated at the apical membrane of acinar cells in the submandibular and parotid glands after administration of isoproterenol, but not pilocarpine. Furthermore, both AQP5 and AQP5-T259A were constitutively localized at the plasma membrane in HSG cells under the resting and forskolin-stimulated conditions. These results suggest that AQP5 is phosphorylated at its Thr259 by PKA through cAMP, but not Ca(2+), signaling pathways, and that this phosphorylation does not contribute to AQP5 trafficking in the salivary gland cells.

American Journal of Physiology, Cell Physiology


Induction of Sca-1 in the duct cells of the mouse submandibular gland by obstruction of the main excretory duct

The effect of ligation of the main excretory duct (MED) of the mouse submandibular gland (SMG) on the expression of Sca-1, a stem cell antigen, was examined by Western blotting and immunohistochemistry. By Western blotting, the expression of Sca-1 with a molecular weight of 18 kDa was identified in the normal gland. At 1 day post-ligation, the expression level of Sca-1 was strongly increased in the experimental gland and weakly in the contralateral gland, and such expression in both glands decreased at 6 days. By immunohistochemistry, Sca-1 was detected weakly in the apical membrane of excretory duct (ED) cells of the SMG under the normal condition. By duct ligation, Sca-1 became expressed strongly in most cells of the two major duct systems, i.e., the striated duct (SD) and granular convoluted tubules (GCT), but was not detected in the acinar (Ac) cells. By fluorescence-activated cell sorter (FACS) analysis, the number of side population (SP) cells in this gland was found to be increased by ligation. These results imply that Sca-1-positive cells may have a role in the duct cell proliferation in the regeneration step elicited by MED ligation-induced injury.


Effects of naturally occurring G103D point mutation of AQP5 on its water permeability, trafficking, and cellular localization in the submandibular gland of rats

AQPs (aquaporins) are water channel proteins that are expressed in almost all living things. In mammalians, 13 members of AQPs (AQP0-12) have been identified so far. AQP5 is known to be expressed mostly in the exocrine cells, including the salivary gland acinar cells. A naturally occurring point mutation (G308A, Gly103 > Asp103) was earlier found in the rat AQP5 gene [Murdiastuti, Purwanti, Karabasil, Li, Yao, Akamatsu, Kanamori and Hosoi (2006) Am. J. Physiol. 291, G1081-G1088]; in this mutant, the rate of initial saliva secretion under stimulated and unstimulated conditions is less than that for the wt (wild-type) animals. Here the mutant molecule was characterized in detail. Using the Xenopus oocyte system, we demonstrated the mutant AQP5 to have water permeability almost the same as that of the wt molecule. Mutant and wt AQP5s, tagged with GFP (green fluorescent protein; GFP-AQP5s) and expressed in polarized MDCK-II (Madin-Darby canine kidney II) cells, first appeared in the vesicular structure(s) in the cytoplasm, and were translocated to the upper plasma membrane or apical membrane during cultivation, with the mutant GFP-AQP5 being translocated less efficiently. Thapsigargin and H-89 both induced translocation in vitro of either molecule, whereas colchicine inhibited this activity; the fraction of cells showing apical localization of mutant GFP-AQP5 was less than that showing that of the wt molecule under any of the experimental conditions used. In the mutant SMG (submandibular gland) tissue, localization of AQP5 in the apical membrane of acinar cells was extremely reduced. Vesicular structures positive for AQP5 and present in the cytoplasm of the acinar cells were co-localized with LAMP2 (lysosome-associated membrane protein 2) or cathepsin D in the mutant gland, whereas such co-localizations were very rare in the wt gland, suggesting that the mutant molecules largely entered lysosomes for degradation. Replacement of highly conserved hydrophobic Gly103 with strongly hydrophilic Asp103 in rat AQP5, though it did not affect water permeability, may possibly have resulted in less efficient membrane trafficking and increased lysosomal degradation, leading to its lower expression in the apical membrane of the acinar cells in the SMG.


Roles of lysosomal proteolytic systems in AQP5 degradation in the submandibular gland of rats following chorda tympani parasympathetic denervation

Chorda tympani denervation (CTD) of rats was earlier shown to result in loss of submandibular gland (SMG) weight (at only 1 wk) and in continued reduction in aquaporin 5 (AQP5) protein expression (until 4 wk), without affecting its mRNA synthesis (Li X, Azlina A, Karabasil MR, Purwanti N, Hasegawa T, Yao C, Akamatsu T, Hosoi K. Am J Physiol Gastrointest Liver Physiol 295: G112-G123, 2008). The present study indicated that despite elevation of bax, a proapoptosis protein, by CTD, the operation also increased the level of bcl-2, an antiapoptosis protein, in the SMG. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL assay) showed no increase in the number of apoptotic cells in the SMG. CTD, however, induced strongly and transiently (at 1-3 days) the protein expression of LC3B-II, a marker protein of autophagosomes, suggesting that the reduction in the gland weight was due to onset of autophagy by CTD. Upon CTD, Lamp2, a lysosomal marker, gradually increased in amount, reaching a peak at the 14th day. Immunohistochemical analysis revealed an increase in the number of lysosome-like structures positive for both AQP5 and Lamp2 in the acinar cells of the SMG after CTD; similar changes were observed also for AQP5 and LC3Bs. These data suggest that AQP5 in the SMG entered autophagosomes and/or lysosomes for degradation upon CTD. In vitro AQP5-degrading activity was found in the SMG extracts, and such activity was shown to be increased by CTD. Inhibitor experiments implied cathepsins B and L to be candidate enzymes for this degradation under normal and CTD conditions, respectively.


Potential down-regulation of salivary gland AQP5 by LPS via cross-coupling of NF-κB and p-c-Jun/c-Fos

The mRNA and protein levels of aquaporin (AQP)5 in the parotid gland were found to be potentially decreased by lipopolysaccharide (LPS) in vivo in C3H/HeN mice, but only weakly in C3H/HeJ, a TLR4 mutant mouse strain. In the LPS-injected mice, pilocarpine-stimulated saliva production was reduced by more than 50%. In a tissue culture system, the LPS-induced decrease in the AQP5 mRNA level was blocked completely by pyrrolidine dithiocarbamate, MG132, tyrphostin AG126, SP600125, and partially by SB203580, which are inhibitors for IkappaB kinase, 26S proteasome, ERK1/2, JNK, and p38 MAPK, respectively. In contrast, the expression of AQP1 mRNA was down-regulated by LPS and such down-regulation was blocked only by SP600125. The transcription factors NF-kappaB (p65 subunit), p-c-Jun, and c-Fos were increased by LPS given in vivo, whereas the protein-binding activities of the parotid gland extract toward the sequences for NF-kappaB but not AP-1-responsive elements present at the promoter region of the AQP5 gene were increased by LPS injection. Co-immunoprecipitation by using antibody columns suggested the physical association of the three transcription factors. These results suggest that LPS-induced potential down-regulation of expression of AQP5 mRNA in the parotid gland is mediated via a complex(es) of these two classes of transcription factors, NF-kappaB and p-c-Jun/c-Fos.


Correlation between aquaporin and water permeability in response to vasotocin, hydrin and {beta}-adrenergic effectors in the ventral pelvic skin of the tree frog Hyla japonica.

The ventral pelvic skin of the tree frog Hyla japonica expresses two kinds of arginine vasotocin (AVT)-stimulated aquaporins (AQP-h2 and AQP-h3), which affect the capacity of the frog's skin to absorb water. As such, it can be used as a model system for analyzing the molecular mechanisms of water permeability. We investigated AQP dynamics and water permeability in the pelvic skin of H. japonica following challenge with AVT, hydrins (intermediate peptides of pro-AVT) and beta-adrenergic effectors. In the in vivo experiment, both AQP-h2 and AQP-h3 proteins were translocated to the apical plasma membrane in the principal cells of the first-reacting cell (FRC) layer in the pelvic skin following challenge with AVT, hydrin 1 and hydrin 2, thereby increasing the water permeability of the pelvic skin. The beta-adrenergic receptor agonist isoproterenol (IP) and its anatagonist propranolol (PP) in combination with AVT or hydrins were used as challenge in the in vitro experiment. IP increased water permeability whereas PP inhibited it, and both events were well correlated with the translocation of the AQPs to the apical membrane. In the PP+AVT-treated skins, labels for AQP-h2 and AQP-h3 were differentially visible among the principal cells; the apical plasma membrane of some cells was labeled while others were not, indicating that the response of PP or AVT is different from cell to cell. These results provide morphological evidence that the principal cells of the FRC layers may have two kinds of receptors: a V2 receptor and beta-adrenergic receptor.


Water adaptation strategy in anuran amphibians: molecular diversity of aquaporin.

Most adult anuran amphibians except for the aquatic species absorb water across the ventral pelvic skin and reabsorb it from urine in the urinary bladder. Many terrestrial and arboreal species use a region in the posterior or pelvic region of the ventral skin that is specialized for rapid rehydration from shallow water sources or moist substrates. Periods of terrestrial activity can be prolonged by reabsorption of dilute urine from the urinary bladder. Aquaporin (AQP), a water channel protein, plays a fundamental role in these water absorption/reabsorption processes, which are regulated by antidiuretic hormone. Characterization of AQPs from various anurans revealed that the unique water homeostasis is basically mediated by two types of anuran-specific AQPs, i.e. ventral pelvic skin and urinary bladder type, respectively. The bladder-type AQP is further expressed in the pelvic skin of terrestrial and arboreal species, together with the pelvic skin-type AQP. In contrast, the pelvic skin-type AQP (AQP-x3) of the aquatic Xenopus has lost the ability of efficient protein production. The extra C-terminal tail in AQP-x3 consisting of 33 nucleotides within the coding region appears to participate in the posttranscriptional regulation of AQP-x3 gene expression by attenuating protein expression. The positive transcriptional regulation of bladder-type AQP in the pelvic skin and negative posttranscriptional regulation of pelvic skin-type AQP provide flexibility in the water regulation mechanisms, which might have contributed to the evolutionary adaptation of anurans to a wide variety of water environments.


Trafficking of GFP-AQP5 chimeric proteins conferred with unphosphorylated amino acids at their PKA-target motif (152SRRTS) in MDCK-II cells

Three constructs having mutated PKA-target motif at (152)SRRTS of AQP5, an exocrine type water channel, were prepared and fused to C-terminus of green fluorescence protein cDNA to examine the effects of blocking of phosphorylation at (152)SRRTS (a consensus PKA-target motif of AQP5) on translocation or trafficking of the chimeric proteins expressed in the Madin-Darby canine kidney-II (MDCK-II) cells. H-89 treatment increased translocation of wild-type GFP-AQP5 to the apical membrane. All 3 mutant molecules translocated 1.5 to 2 times more than the control wild-type GFP-AQP5. Colchicine but not cytochalasin B inhibited the translocation of wild-type GFP-AQP5. Present results suggest dephosphorylation of this consensus sequence increase GFP-AQP5 translocation, and that microtubules but not microfilaments are involved in this event.

The Journal of Medical Investigation : JMI


Inhibition and transcriptional silencing of a subtilisin-like proprotein convertase, PACE4/SPC4, reduces the branching morphogenesis of and AQP5 expression in rat embryonic submandibular gland

The submandibular gland (SMG) develops through the epithelial-mesenchymal interaction mediated by many growth/differentiation factors including activin and BMPs, which are synthesized as inactive precursors and activated by subtilisin-like proprotein convertases (SPC) following cleavage at their R-X-K/R-R site. Here, we found that Dec-RVKR-CMK, a potent inhibitor of SPC, inhibited the branching morphogenesis of the rat embryonic SMG, and caused low expression of a water channel AQP5, in an organ culture system. Dec-RVKR-CMK also decreased the expression of PACE4, a SPC member, but not furin, another SPC member, suggesting the involvement of PACE4 in the SMG development. Heparin, which is known to translocate PACE4 in the extracellular matrix into the medium, and an antibody specific for the catalytic domain of PACE4, both reduced the branching morphogenesis and AQP5 expression in the SMG. The inhibitory effects of Dec-RVKR-CMK were partially rescued by the addition of recombinant BMP2, whose precursor is one of the candidate substrates for PACE4 in vivo. Further, the suppression of PACE4 expression by siRNAs resulted in decreased expression of AQP5 and inhibition of the branching morphogenesis in the present organ culture system. These observations suggest that PACE4 regulates the SMG development via the activation of some growth/differentiation factors.


Degradation of submandibular gland AQP5 by parasympathetic denervation of chorda tympani and its recovery by cevimeline, an M3 muscarinic agonist

By chorda tympani denervation (CTD, parasympathectomy), the aquaporin 5 (AQP5), but not AQP1, protein level in the rat submandibular gland (SMG) was significantly decreased, dropping to 37% of that of the contralateral gland at 4 wk. The protein levels of AQP5 and AQP1 were not significantly affected by denervation of the cervical sympathetic trunk (sympathectomy). Administration of cevimeline hydrochloride, an M3 muscarinic receptor agonist (10 mg/kg for 7 days po), but not pilocarpine (0.3 mg/kg for 7 days po), recovered the AQP5 protein level reduced by CTD and increased the AQP1 protein level above the control one. The mRNA level of AQP5 was scarcely affected by CTD and cevimeline hydrochloride administration. Administration of chloroquine (50 mg/kg for 7 days po), a denaturant of lysosomes, increased the AQP5 protein level reduced by CTD. An extract obtained from the submandibular lysosomal fraction degraded the AQP5 protein in the total membrane fraction in vitro. These results suggest the possible regulation of the AQP5 protein level in the SMG by the parasympathetic nerves/M3 muscarinic receptor agonist and imply the involvement of lysosomal enzymes, but not a transcriptional mechanism, in this regulation.

American Journal of Physiology, Gastrointestinal and Liver Physiology


Gene cloning and expression of an aquaporin (AQP-h3BL) in the basolateral membrane of water-permeable epithelial cells in osmoregulatory organs of the tree frog.

An aquaporin (Hyla AQP-h3BL), consisting of 292 amino acid residues, has been cloned from the urinary bladder of Hyla japonica. In a swelling assay using Xenopus oocytes, AQP-h3BL cRNA-injected oocytes developed a sevenfold and 2.8-fold higher permeability to water and glycerol, respectively, than the water-injected oocytes. This permeability was inhibited by HgCl2. Immunofluorescence revealed that AQP-h3BL is localized in the basolateral plasma membrane of both granular cells in the ventral pelvic and dorsal skins and the secretory cells in the mucous glands. Immunopositive cells were also observed in the basolateral membrane of principal cells in the collecting ducts and in a portion of the late distal tubules in the kidneys, as well as in the principal cells of the urinary bladder. Sequence homology suggests that AQP-h3BL is a homolog to mammalian AQP3. This conclusion is supported by the observed localization of AQP-h3BL to the basolateral membrane in water- and glycerol-permeable epithelial cells. In ventral pelvic skins and urinary bladders, water enters into the cytoplasm through the apical plasma membrane at sites where AQP-h2, sometimes in association with AQP-h3, responds to stimulation by vasotocin; the water exits throughout AQP-h3BL to extracellular spaces. In the mucous glands, on the other hand, water enters throughout this AQP-h3BL and exits through AQP-x5, which is in the apical membrane of secretory cells. Thus, water homeostasis in the frog body is regulated by AQP-h3BL expressed in the basolateral membrane in concert with arginine vasotocin (AVT)-dependent or AVT-independent AQP.

American Journal of Physiology. Regulatory, Integrative and Comparative Physiology


Differential localization of aquaporin-2 and glucose transporter 4 in polarized MDCK cells.

Membrane water channel aquaporin-2 (AQP2) and glucose transporter 4 (GLUT4) exhibit a common feature in that they are stored in intracellular storage compartments and undergo translocation to the plasma membrane upon hormonal stimulation. We compared the intracellular localization and trafficking of AQP2 and GLUT4 in polarized Madin-Darby canine kidney cells stably transfected with human AQP2 (MDCK-hAQP2) by immunofluorescence microscopy. When expressed in MDCK-hAQP2 cells, GLUT4 and GLUT4-EGFP were predominantly localized in the perinuclear region close to and within the Golgi apparatus, similar to endogenous GLUT4 in adipocytes and myocytes. In addition, GLUT4 was occasionally seen in EEA1-positive early endosomes. AQP2, on the other hand, was sequestered in subapical Rab11-positive vesicles. In the basal state, the intracellular storage site of GLUT4 was distinct from that of AQP2. Forskolin induced translocation of AQP2 from the subapical storage vesicles to the apical plasma membrane, which did not affect GLUT4 localization. When forskolin was washed out, AQP2 was first retrieved to early endosomes from the apical plasma membrane, where it was partly colocalized with GLUT4. AQP2 was then transferred to Rab11-positive storage vesicles. These results show that AQP2 and GLUT4 share a common compartment after retrieval from the plasma membrane, but their storage compartments are distinct from each other in polarized MDCK-hAQP2 cells.


Cloning and expression of vacuolar proton-pumping ATPase subunits in the follicular epithelium of the bullfrog endolymphatic sac.

In an investigation aimed at clarifying the mechanism of crystal dissolution of the calcium carbonate lattice in otoconia (the mineral particles embedded in the otolithic membrane) of the endolymphatic sac (ELS) of the bullfrog, cDNAs encoding the A- and E-subunits of bullfrog vacuolar proton-pumping ATPase (V-ATPase) were cloned and sequenced. The cDNA of the A-subunit consisted of an 11-bp 5'-untranslated region (UTR), a 1,854-bp open reading frame (ORF) encoding a protein comprising 617 amino acids with a calculated molecular mass of 68,168 Da, and a 248-bp 3'-UTR followed by a poly(A) tail. The cDNA of the E-subunit consisted of a 72-bp 5'-UTR, a 681-bp ORF encoding a protein of 226 amino acids with a calculated molecular mass of 26,020 Da, and a 799-bp 3'-UTR followed by a poly(A) tail. Western blot and immunofluorescence analyses using specific anti-peptide antisera against the V-ATPase A- and E-subunits revealed that these subunits were present in the ELS, urinary bladder, skin, testes, and kidneys. In the ELS, positive cells were scattered in the follicular epithelium which, as revealed by electron microscopy, corresponds to the location of mitochondria-rich cells. These findings suggest that V-ATPase, including the A- and E-subunits, exists in mitochondria-rich cells of the ELS, which might be involved in dissolution of the calcium carbonate crystals in the lumen of the ELS.


Molecular and cellular characterization of a new aquaporin, AQP-x5, specifically expressed in the small granular glands of Xenopus skin.

A new toad aquaporin (AQP) cDNA was cloned from a cDNA library constructed from the ventral skin of Xenopus laevis. This AQP (Xenopus AQP-x5) consisted of 273 amino acid residues with a high sequence homology to mammalian AQP5. The predicted amino acid sequence contained the two conserved Asn-Pro-Ala motifs found in all major intrinsic protein (MIP) family members and six putative transmembrane domains. The sequence also contained a mercurial-sensitive cysteine and a putative phosphorylation motif site for protein kinase A at Ser-257. The swelling assay using Xenopus oocytes revealed that AQP-x5 facilitated water permeability. Expression of AQP-x5 mRNA was restricted to the skin, brain, lungs and testes. Immunofluorescence and immunoelectron microscopical studies using an anti-peptide antibody (ST-156) against the C-terminal region of the AQP-x5 protein revealed the presence of immunopositive cells in the skin, with the label predominately localized in the apical plasma membrane of the secretory cells of the small granular glands. These glands are unique both in being close to the epidermal layer of the skin and in containing mitochondria-rich cells with vacuolar H+-ATPase dispersed among its secretory cells. Results from immunohistochemical experiments on the mucous or seromucous glands of several other anurans verified this result. We conclude that the presence of AQP-x5 in the apical plasma membrane of the small granular glands suggests its involvement in water secretion from the skins. The physiological roles of the AQP-x5 protein in the small or mucous glands are discussed.


A starvation-specific serine protease gene, isp6+, is involved in both autophagy and sexual development in Schizosaccharomyces pombe.

Schizosaccharomyces pombe isp6(+) gene encodes a vacuolar serine protease, which is specifically induced during nitrogen starvation. An isp6-disruption mutant, isp6Delta, grew normally under normal conditions but was defective in large-scale protein degradation during nitrogen starvation, a hallmark of autophagy. Vacuoles are the organelles for such drastic protein degradation but those of isp6Delta were apparently aberrant. isp6Delta was infertile under nitrogen source-free conditions with poor expression of ste11(+), a gene critical for sexual development. A protein kinase A-disruption mutant, pka1Delta, is prone to sexual development because expression of ste11(+) is derepressed. However, isp6Deltapka1Delta still showed defects in ste11(+) expression and sexual development under nitrogen source-free conditions. isp6Delta and isp6Deltapka1Delta were able to initiate sexual development to produce spores when only a small amount of a nitrogen source was present. Pat1 protein kinase negatively controls meiosis, and a temperature-sensitive mutant of pat1, pat1-114, initiates meiosis irrespective of ploidy at the restrictive temperature. However, isp6Deltapat1-114 did not start meiosis under nitrogen source-free conditions even at the restrictive temperature. These observations suggest that isp6(+) contributes to sexual development by providing a nitrogen source through autophagy.


Immunocytochemical studies on translocation of phosphorylated aquaporin-h2 protein in granular cells of the frog urinary bladder before and after stimulation with vasotocin.

We have generated a specific antibody against phosphorylated aquaporin-h2 (pAQP-h2) protein to investigate the role of phosphorylation in the translocation of AQP-h2 protein within the granule cells of the urinary bladder of the frog (Hyla japonica). The antibody was generated against a synthetic peptide (ST-160) corresponding to amino acids 255-268, with a phosphorylated Ser-262, a residue that is putatively phosphorylated by protein A kinase. Using this antibody, we found, by Western blot analysis, that phosphorylation of the AQP-h2 protein rapidly increased within 2 min after vasotocin (AVT) stimulation and remained at a higher than normal level for 15 min. Moreover, quantitative immunoelectron microscopy indicated that the location of the AQP-h2 protein dramatically changed after AVT stimulation. Before stimulation, pAQP-h2 protein was localized in only a small number of intracellular vesicles near the nucleus of the granular cells, whereas the labeling density of the intracellular vesicles and the apical membrane rapidly increased after stimulation. This finding was also confirmed by the results of an immunofluorescence study. Thus, phosphorylation of AQP-h2 protein seems to be essential for translocation of the protein from the cytoplasmic pool to the apical plasma membrane of the granular cells in frog urinary bladder.


Immunocytochemical and phylogenetic distribution of aquaporins in the frog ventral skin and urinary bladder.

We recently cloned three cDNAs encoding frog aquaporin (AQP-h1, BAC07470; AQP-h2, BAC82379; and AQP-h3, BAC07471) from the ventral pelvic skin of the tree frog, Hyla japonica. The present study demonstrated that Hyla AQP-h2 was translocated from cytoplasmic pools to the apical plasma membranes of the granular cells in the bladder after antidiuretic hormone stimulation and that Hyla AQP-h2 and AQP-h3 behaved similarly in the ventral pelvic skin. Further, we found that terrestrial and tree frogs, but not aquatic and semiterrestrial-adapted frogs, absorbed water from their ventral pelvic skin by AQP-h3-like protein in concert with AQP-h2-like protein.

Annals of the New York Academy of Sciences


Spatial and temporal expression of the ventral pelvic skin aquaporins during metamorphosis of the tree frog, Hyla japonica.

Most adult anurans absorb water through their ventral skin to maintain the proper water balance. We examined spatial and temporal expression of frog (Hyla japonica) aquaporins, Hyla AQP-h2 and AQP-h3 proteins, in the ventral pelvic skin by using specific antibodies. Immunofluorescence indicates that AQP-h2 and AQP-h3 first appear in the granular cells of the pelvic skin of the tadpoles at Gosner stage 42, and such labeling is seen in later stages as well. These findings were confirmed by Western blot analysis. In addition, Northern blot analysis demonstrated that V2-type vasotocin (AVT)-receptor mRNA is first expressed at the same stage as are the AQP proteins, which suggests a functional relationship between expression of AQP proteins and AVT receptor. Also, AQP expression in the ventral pelvic skin is consistent with the morphological changes that occur in the skin for adaptation from life in water to that on land.


Regulation of water absorption in the frog skins by two vasotocin-dependent water-channel aquaporins, AQP-h2 and AQP-h3.

A new frog aquaporin (AQP) cDNA was cloned from a cDNA library constructed from the ventral skin of the tree frog Hyla japonica. This AQP (Hyla AQP-h2) consisted of 268 amino acid residues with a high homology to mammalian AQP2. The predicted amino acid sequence contained the two conserved Asn-Pro-Ala motifs found in all the major intrinsic protein family members and the putative six transmembrane domains. The sequence also contained a mercurial compound: cysteine, one potential N-glycosylation site at Asn-124, and a putative phosphorylation site recognized by protein kinase A at Ser-262. In a swelling assay using Xenopus oocytes, AQP-h2 facilitated water permeability, especially in response to cAMP. Expression of AQP-h2 mRNA was restricted to several tissues including the ventral skin, kidney, and urinary bladder; but with immunofluorescence staining using an antipeptide antibody (ST-140) against the AQP-h2 protein, immunopositive cells were found only in the ventral skin and urinary bladder. In the ventral pelvic skin, the label for AQP-h2 was localized in the entire plasma membrane of the granular cells beneath the outmost layer of the skin and in the basolateral membrane of the granular cells in this layer. In response to vasotocin, however, the label for AQP-h2 became more intense in the apical membrane in the granular cells of the outermost layer, similar to the case for the earlier studied AQP-h3, which was specifically expressed in the ventral skin. Taken together, these findings suggest that not only AQP-h3, but also AQP-h2 acts as a regulator of the water balance in this frog.


Chloroplasts in seeds and dark-grown seedlings of lotus.

In most higher plants, mature dry seeds have no chloroplasts but etioplasts. Here we show that in a hydrophyte, lotus (Nelumbo nucifera), young chloroplasts already exist in shoots of mature dry seeds and that they give rise to mature chloroplasts during germination, even in darkness. These shoots contain chlorophyll and chlorophyll-binding proteins CP1 and LHCP. The unique features of chloroplast formation in N. nucifera suggest a unique adaptive strategy for seedling development correlated with the plant's habitat.


Molecular and cellular characterization of a water-channel protein, AQP-h3, specifically expressed in the frog ventral skin.

Two cDNAs encoding frog aquaporin (AQP) were cloned from a cDNA library constructed for the ventral skin of the tree frog, Hyla japonica and sequenced. One AQP (Hyla AQP-h1) consisted of 271 amino-acid residues with high homology to toad AQP-t1, Rana CHIP28 (AQP1), and rat AQP1. The other AQP (AQP-h3) consisted of 271 amino-acid residues with higher homology to mammalian AQP2 than to mammalian AQP3. The predicted amino-acid sequence contained the conserved two NPA motifs found in all MIP family members and the putative six transmembrane domains. The sequence also confers mercurial sensitivity, which is common to all the AQPs except AQP0, AQP4 and AQP7. Potential N-glycosylation sites were present at Asn-44 in AQP-h1, and at Asn-124 and Asn-125 in AQP-h3. In addition, AQP-h3 had a putative phosphorylation site by protein kinase A at Ser-255, which is identical to mammalian AQP2. In swelling assays using Xenopus oocytes, AQP-h1 facilitates water permeability, whereas AQP-h3 displayed weak water permeability. Searching for the expression of these two AQP mRNAs revealed that AQP-h1 was expressed in most tissues, whereas AQP-h3 was observed only in the ventral skin. An antibody (ST-141) against the C-terminal peptide of the AQP-h3 protein recognized a 29.0 kDa-protein with a molecular mass close to that of the Hyla AQP-h3 protein and immunostained predominantly in the abdominal pelvic skin. In pelvic skin, the label for AQP-h3 was more intense in the upper layer of the stratum granulosum and was localized to both the apical and basolateral plasma membranes of the principal cells. These findings suggest that Hyla AQP-h3 plays a pivotal role in constitutively absorbing water from ventral pelvic skin.


The Defense System of Oral Cavity: Lipopolysaccharide Induced Inflammatory Response in the Mice Salivary Gland

BIT's 1st Annual World Congress of Oral & Dental Medicine Conference Abstract Book


Salivary Gland Development, Differentiation, and Regeneration - Role of Subtilisin-like Proprotein Convertase PACE4/SPC4


Enhanced ubiquitylation and downregulation of aquaporin-5 by ubiquitin ligases

ユビキチンリガーゼによるAQP5のユビキチン化亢進とダウンレギュレーション


Expression of water channel AQP5 in regeneration model of salivary gland

唾液腺再生モデルにおける水チャネルAQP5の発現


Induced expression of subtilisin-like proprotein convertase in regeneration model of salivary gland

唾液腺再生モデルにおけるサチライシン様前駆体蛋白質変換酵素の発現誘導